We examined the effects of secretin (0 to 200 nM) and caerulein (0 to 100 nM) on rat pancreatic acinar cells cultured 0 to 48 h in serum-free medium. The effects of 100 nM secretin with 1 nM caerulein were also studied because secretin may potentiate the effects of caerulein. Cellular and media (secreted) lipase and amylase were analyzed as were cellular DNA and protein content. Cellular lipase and amylase activities significantly decreased (P less than 0.0001) over time in all treatment groups, whereas media amylase and lipase significantly increased (P less than 0.0001). Neither secretin nor caerulein affected cellular lipase or media amylase. However, secretin significantly increased (P less than 0.04) and caerulein tended to increase (P less than 0.08) media lipase in a dose-dependent manner. At 12 h, 10 nM secretin maximally increased media lipase (58%) suggesting that cultured acinar cells remain responsive to secretin in vitro. Caerulein, at all concentrations, significantly decreased (P less than 0.001) cellular amylase but exhibited a dose-dependent effect only at 24 h when 100 nM caerulein maximally decreased cellular amylase (34%). Secretin (100 nM) did not alter these effects of caerulein. These results support the proposed role of caerulein in the regulation of amylase but not a direct role of secretin in the regulation of lipase.