Rossi takes pole, Hayden on second row
The 27-year-old Italian, bidding for his sixth consecutive world title in the premier class, clocked one minute 31.002 on his Yamaha at the tight and twisty 4km circuit.
Nicky Hayden, the only man who can deprive Rossi of the title, was on the second row of the grid on his Honda.
Ducati duo Troy Bayliss and Loris Capirossi joined Rossi on the front row while Hayden will be accompanied by teammate Dani Pedrosa and Kawasaki rider Shinya Nakano on the second.
Casey Stoner, fellow Australian Chris Vermeulen and John Hopkins are on the third row. Colin Edwards, Randy de Puniet and Marco Melandri make up the fourth.
"Tomorrow (today) is the most important race of the year," Rossi told Spanish television after claiming his fifth pole position of the season.
"I will be fully concentrated from the first to the last minute. The bike is going very well and I feel comfortable."
Rossi grabbed the lead in the title race for the first time this season when he finished second in Portugal earlier this month.
He leads the championship by eight points from Hayden, who crashed out at Estoril after his Honda teammate Pedrosa ploughed into him on a corner.
The Italian will wrap up his eighth world title in all classes if he finishes ahead of the American in today's race. But Hayden can turn the tables if he wins and the Yamaha rider fails to finish second.
Capirossi set the early pace in qualifying on Saturday before his Ducati teammate Bayliss and Hayden beat his mark as the riders used their best qualifying tires.
Rossi pulled out all the stops in the final minutes of the session to beat Sete Gibernau's previous pole record by more than 8/10ths of a second.
World champion Alvaro Bautista claimed his eighth pole of the season in the 125cc class with Aprilia teammates Hector Faubel, Sergio Gadia and Mattia Pasini completing the front row.