Driving with his usual elan and precision in a gusty wind at the Circuit de Catlunya, the two-time world champion clocked a best lap of one minute and 26.852 seconds to finish almost half a second clear of nearest rival four-time champion German Sebastian Vettel of Ferrari.
Rosberg, given a reprimand during the morning for infringing the pit-lane entry lines as he worked his way to the fastest lap in that session, wound up seven-tenths of a second back in third place.
Finn Kimi Raikkonen, who complained of boredom during a radio conversation in the closing stages, was fourth quickest in the second Ferrari, endorsing a growing impression that the Italian team is the only one capable of mounting a serious challenge to Mercedes' continuing domination, especially if it can outperform the champions in tyre-wear management.
For Briton Hamilton, it was another day of business as usual. After winning three of the season-opening four "flyaway" races outside Europe, he dominated the session. He has failed to top the times in second practice only five times in the last 24 sessions.
More From This Section
This meant Grosjean was delayed in the pits for more than half an hour for repairs - a predicament shared by Daniel Ricciardo as the Australian's Red Bull car was up on its stand in the team garage for 55 minutes of the session as his fourth and final penalty-free Renault power unit of the season was installed.
That left him eight places behind his Red Bull team-mate Russian Daniil Kvyat, who was a fine fifth after a lacklustre start to his season ahead of Dutch teenager Max Verstappen, of Toro Rosso.
Briton Jenson Button delivered a welcome shaft of optimism to the troubled McLaren Honda team in seventh place ahead of Finn Valtteri Bottas of Williams, Spanish local hero Carlos Sainz in the second Toro Rosso and Brazilian Felipe Massa in the second Williams.