Francesco Bagnaia began his home Italian MotoGP weekend in style, Ducati's double world champion topping the times in practice on Friday.
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Mugello is one of the Turin-born star's favourite circuits and he came within a tenth of a second of the lap record he set last year.
With his best time of 1min 44.938sec he led the way from Spanish duo Alex Rins (Yamaha) and rookie Pedro Acosta (GasGas).
Six-time former champion Marc Marquez posted the fifth fastest time with series leader Jorge Martin in seventh.
The top 10 riders booked their place in Saturday's second qualifying session.
This group, which determines the first four rows of the grid, are joined by the two fastest riders from Q1.
Ahead of Saturday's sprint and Sunday's main event Bagnaia is lying second in the riders' standings, 39 points behind Martin.
Marquez is placed third, two points behind Bagnaia, before this seventh round of the season.
Bagnaia, winner last weekend in Catalunya, is seeking a third straight win in front of tens of thousands of his tifosi supporters in Sunday's race.