Calcium is an important mediator of pancreatic stimulus-secretion coupling, and in streptolysin O-permeabilized acini maximal amylase release can be induced by 1 microMCa2+. Two-dimensional analysis of acinar phosphoproteins using 32P-labeled acini showed that 1-min stimulation of permeabilized acini with 1 microMCa2+ induced a number of rapid protein phosphorylation and dephosphorylation events similar to previously observed effects of secretagogues acting on intact acini. The rapid Ca2+ effects on protein phosphorylation correlated well with the earliest detectable onset of amylase release after 1 min of incubation with Ca2+. Our findings indicate that some of the agonist-induced changes in protein phosphorylation are mediated by Ca2+ and also provide evidence for the possible involvement of Ca(2+)-mediated dephosphorylation in acinar stimulus-secretion coupling.