Pancreastatin (PST), a peptide isolated from porcine pancreas in 1986, has been reported to inhibit insulin and to stimulate glucagon secretion. Since both of these effects have been questioned, we investigated the effect of PST (20, 200, or 2000 pM) on hormone release in the isolated perfused rat pancreas at different glucose levels (1.7, 5.5, 11.1, and 16.7 mM). At 1.7 mM glucose, 20 pM PST had no significant effect on glucagon secretion, whereas 200 pM and 2 nM PST significantly inhibited glucagon release. At a concentration of 5.5 mM glucose, insulin output was not affected by PST in any of the concentrations tested. At 11.1 mM glucose, however, 200 pM and 2 nM PST significantly inhibited insulin output. At 16.7 mM glucose, insulin secretion was significantly reduced by all concentrations of PST tested. Unstimulated exocrine pancreatic secretion was not affected by PST in any of the experimental settings. We conclude that PST inhibits glucagon and insulin secretion dose-dependently, and these effects apparently are glucose-dependent. PST does not influence basal exocrine pancreatic secretion in vitro.