To define the hormonal regulation of the exocrine pancreas, acini were maintained for 24 h in suspension culture under various conditions and then examined morphologically and tested for their ability to synthesize and secrete protein. Acini cultured in the presence of 20% fetal calf serum (control conditions) retained their differentiated morphology as judged by light and electron microscopy. Under control conditions, stimulation with either cholecystokinin octapeptide (CCK8) or carbachol increased the release of amylase in a dose-dependent manner with a maximal threefold increase occurring at 200 pM CCK8 or 6 microM carbachol. Without inclusion of other factors in the medium during the culture period, the rate of protein synthesis measured in cultured acini was one-third the rate measured in freshly prepared acini. Inclusion of epidermal growth factor (EGF) (2 nM), carbachol (1 microM), or insulin (1 microM) during the culture period led to increases, whereas corticosterone (1 microM) led to decreases in functional parameters. Greatest effects were seen with a combination of EGF, carbachol, and insulin. This combination of factors led to a 160% increase over control in CCK8-stimulated amylase release and a 180% increase in [3H]leucine incorporation. These results indicate that isolated pancreatic acini can be maintained for up to 24 h and that EGF, carbachol, insulin, and corticosterone can regulate the long-term function of this tissue.