We have measured protein phosphatase (PP) activity in crude homogenates as well as in the total 105.000 x g supernatant and precipitate fractions from normal rat pancreatic islets. On the basis of the inhibition produced by either 1 nM or 1 microM okadaic acid, both PP1 and PP2A activity were present in crude islet homogenates in equivalent proportions (53% and 47%, respectively); PP1 was the main activity present in the precipitate, whereas in the supernatant it was PP2A. Tolbutamide, glybenclamide and glyclazide significantly decreased PP activity in islet homogenates in a dose-dependent manner, with a K10.5 value that in the case of glybenclamide correlated with its Kd for binding site, its EC50 on KATP channel, and its EC50 on insulin release. These data indicate that PPs play a role in the control of insulin secretion and suggest a further possible target for sulfonylureas within their overall action as insulin secretagogues.