Sphingosine and ceramide, products of sphingomyelin hydrosis by sphingomyelinase, have recently been regarded as second messengers for cell biological actions. On the other hand, exocrine pancreas is a typical organ to perform regulatory secretion of digestive enzymes, depending upon extracellular signals. We investigated the effects of sphingosine or ceramide on amylase secretion from isolated rat pancreatic acini. Either sphingosine or cell-permeable ceramide inhibits CCK8- or carbachol-induced enzyme secretion from isolated rat pancreatic acini in a dose-dependent manner. Sphingosine or ceramide itself does not affect basal amylase secretion from the acini. Ceramide also inhibits NaF-induced amylase secretion, indicating that it acts post the activation of receptor-linked GTP-binding protein. In our experiments, ceramide inhibited Ca2+ ionophore-induced amylase secretion, but not phorbol ester-induced secretion. These results indicate that ceramide affects secretory processes post intracellular Ca2+ mobilization in the exocrine pancreas.