Speaking in parliament, Mr Rajoy said Carles Puigdemont has until next Monday morning (GMT) "to say if he declared independence" before suspending Catalan autonomy.
"It is very important that Mr Puigdemont clarify for the rest of Spaniards if he declared independence yesterday or not," Mr Rajoy said.
If Puigdemont confirms his region had split from Spain, the central government would give him an additional five days, to October 19 at 10 am (GMT), to reconsider before suspending Catalan autonomy, Mr Rajoy added.
Mr Puigdemont announced in the regional parliament yesterday that he had accepted the mandate for "Catalonia to become an independent state" following the contested referendum.
But in a parliamentary speech Puigdemont immediately called for independence to be suspended, to allow for negotiations with Madrid.
Prime Minister Rajoy has taken the first step towards activating article 155 of the Spanish constitution, a so-called nuclear option that would allow him to suspend Catalonia's political autonomy and take over the region.
Under Article 155, the central government could take control of the governance of a region, a never-before used provision that has been referred to as the "nuclear option".
"The answer from the Catalan president will determine future events, in the next few days," Mr Rajoy said in a televised address.
"The government wants to offer certainty to Spaniards, especially Catalans, it wants to avoid the confusion that has been generated by Catalan authorities."
If Catalan leader Carles Puigdemont reestablishes "institutional normality" this would "put an end to a period of instability and tension."
'Doesn't know where he's going'
At stake is the future of a region of 7.5 million people deeply divided over independence, one of Spain's economic powerhouses whose drive to break away has raised concern for stability in the European Union.
Crowds of thousands gathered outside the parliament building in Barcelona on Tuesday evening, waving Catalan flags and banners screaming "democracy" in the hope of witnessing a historic night in a region that remains deeply divided over independence.
But Spain's political establishment rounded on Mr Puigdemont following his declaration and support among separatists in Catalonia was mixed.
"Mr Puigdemont, no one, can expect to impose mediation without returning to legality or democracy," Deputy Prime Minister Soraya Saenz de Santamaria told reporters.
She said Puigdemont was "a person who doesn't know where he is, where he's going or with whom he wants to go".
Barcelona resident Maria Rosa Bertran said she was against a delayed secession.
"I find it even worse because it is suffering a longer agony. Indecision and uncertainty is the worst thing that can happen to us," she told AFP.
Unknown consequences
Following his declaration to parliament, Mr Puigdemont and his allies signed an independence declaration outside the chamber, but its legal validity was unclear.
Catalonia pressed ahead with an independence referendum on October 1 that the central government said breached Spain's constitution.
Spanish police cracked down on the polls, beating some voters as they closed down polling stations.
"I did not expect independence to be declared today because of all the processes that the government of Spain has begun, both with police actions and with threats," Marc Cazes, a student in Barcelona, said on Tuesday.
About 90 per cent of those who cast ballots voted for independence but the poll was poorly monitored and many Catalans opposed to secession boycotted it.
The crisis has caused deep uncertainty for businesses in one of the wealthiest regions in the eurozone's fourth-largest economy.
A string of companies have already moved their legal headquarters, but not their employees, from Catalonia to other parts of the country.
But on Wednesday morning, the Spanish stockmarket was up 1.16 percent on hopes for a breakthrough in the crisis.
The stand-off has also put strain on the euro.
The single currency was up after Mr Puigdemont's announcement and held onto gains in Asian trade on Wednesday, buying $1.1815.
But it was still down two cents from its recent highs seen last month due to the political uncertainty.
Stephen Innes, head of Asia-Pacific trading at OANDA, said while the euro rose it "gained little traction as this is little more than kicking the can down the road. It's unlikely we've heard the last of this debate despite cooler heads prevailing".
Demands for independence in Catalonia, which has its own language and cultural traditions, date back centuries.
But a 2010 move by Spain's Constitutional Court to water down a statute that gave Catalonia additional powers, combined with a deep economic meltdown in Spain, sparked a surge in support for independence.
