Caerulein is proposed to regulate the synthesis of pancreatic proteases and amylase. Similarly, secretin is implicated in the regulation of pancreatic lipase synthesis. Evidence of these regulations is predominantly from in vivo studies. We therefore examined the effects of caerulein and secretin directly on acinar cells to eliminate possible interactions with other regulatory factors. Cellular and media enzyme activities and relative synthesis were measured after 24 h of hormonal treatment. Cells were incubated with [14C]-amino acids and then subjected to two-dimensional gel electrophoresis to separate individual acinar proteins for subsequent determination of incorporated radioactivity and relative synthesis. In general, all enzyme activities decreased (33%; p < 0.02) over time in culture and medium enzyme activities increased (370%; p < 0.00001) in all treatment groups. Caerulein further decreased cellular content of all enzymes (p < 0.002) and increased media amylase and lipase activities (p < 0.02). Caerulein, however, significantly increased the relative synthesis of trypsin (28%) and tended to increase that of chymotrypsin (25%; p < 0.06), which supports its proposed role in protease regulation. Secretin, on the other hand, did not significantly affect the cellular or medium activities or the relative synthesis of any pancreatic enzyme evaluated. Therefore, this study does not support the proposed role of secretin in lipase regulation.