Cholecystokinin (CCK) is known to rapidly and transiently increase both [Ca2+]i and autonomous CaM kinase II activity in rat pancreatic acini. Because induction of autonomous activity may involve intramolecular autophosphorylation, the effects of protein phosphatase inhibitors were examined. None of the inhibitors tested (okadaic acid, calyculin A, and cyclosporin A) affected basal activity. Okadaic acid, a potent inhibitor of PP2A and weaker inhibitor of PP1, increased the peak autonomous activity by 30% over the level normally induced by CCK alone, while calyculin A, a potent inhibitor of both PP1 and PP2A, showed an even greater increase of 97%. Both inhibitors also delayed the decline of autonomous activity and calyculin A had a more potent effect than okadaic acid. Cyclosporin A, an inhibitor of PP2B, had no effect. The data indicate that PP1 may be involved in the dephosphorylation of CaMK II and decline of autonomous activity.