Hamilton, whose team risks heavy sanctions after a storm over lying to stewards at the Australian season-opener, lapped the Sakhir circuit in one minute, 33.647 seconds.
The Briton, whose team were disqualified in Australia and have only eight points from three races, had also been quickest in the corresponding session in China last week before being replaced at the top in the afternoon.
BMW-Sauber filled the next two places on the timesheets on a hot and overcast morning with Germany's Nick Heidfeld ahead of Poland's Robert Kubica.
The top three cars were all equipped with the new KERS energy recovery system, that offers drivers an extra boost of power at the push of a button, at a circuit where it is expected to be a real advantage.
Williams' Nico Rosberg, who has so far failed to translate his speed in practice into similarly high-flying race results, was fourth fastest ahead of Brawn GP's championship leader Jenson Button.
Button, who won the first two races of the season and finished third in China, is seen as a favorite to return to the top of the podium.
McLaren has brought new developments to Bahrain to close the gap at the top.
"We have a few modifications for the floor, hopefully a little bit more downforce, but nothing dramatic," said Heikki Kovalainen, whose teammate has been kept well away from the media in Bahrain.
"There is nothing major at this race. I think the next big package should be in Spain."
In the second practice, Rosberg continued his big practice performance. For the seventh time in 11 practice sessions this season, the German topped the time-sheets and will be hoping he can transfer that pace to the GP.
Rosberg went around the Sakhir circuit in 1 minute, 33.339 seconds to finish in front of Renault's Fernando Alonso, Toyota's Jarno Trulli and the Red Bull pair of Sebastian Vettel and Mark Webber, who finished 1-2 in last week's race in China.