The biological activity of phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate, have been associated with activation of phospholipid/Ca²⁺-dependent protein kinase. Treatment of rat pancreatic acini with 12-O-tetradecanoylphorbol-13-acetate (10^-6 m) resulted in a sustained translocation of phospholipid/Ca²⁺-dependent protein kinase activity to the membrane site. The pattern of phosphorylation of at least two substrate proteins (M_r 22,000 and 18,000) for this Ca²⁺-dependent protein kinase was also altered following exposure to phorbol ester, these phosphoproteins disappearing from the soluble fraction and appearing in the particulate. Concurrently, 12-O-tetradecanoylphorbol-13-acetate stimulated amylase release from intact acini in a time- and dose-dependent fashion. These results suggest a potential role for phospholipid/Ca²⁺-activated protein kinase in the regulation of pancreatic exocrine function.