Trifluoperazine, an inhibitor of calcium-calmodulin functions, was used in an attempt to understand the involvement of calcium-calmodulin in glucose-stimulated insulin release. Isolated rat pancreatic islets were used after a two-day period of maintenance in tissue culture. 45Ca2+ uptake and insulin release were measured during 5-min incubations. Dynamic insulin release and 45Ca2+ efflux were assessed during perifusion of the islets preloaded with 45Ca2+ during the culture period. Both phases of insulin release in response to 16.7 mM glucose were inhibited by approximately 60% in the presence of 10 μM trifluoperazine when the latter was added 35 min prior to high glucose. Stimulation of 45Ca2+ efflux by glucose was abolished. Glucose-stimulated 45Ca2+ uptake was inhibited by 43%. These results were compared with those of experiments in which depolarizing concentrations of potassium (24 mM) were used. Trifluoperazine inhibited K+-stimulated insulin release and 45Ca2+ uptake to a similar extent as that seen with glucose. Trifluoperazine did not appear to interfere with the inhibitory effect of glucose on 45Ca2+ efflux seen in the absence of extracellular Ca2+. Moreover, in Ca2+ deprived medium (with no possibility for Ca2+ uptake) insulin release in response to glucose + ouabain or in response to veratridine was also inhibited by trifluoperazine. It can be speculated that calmodulin is involved in the process by which glucose and potassium stimulate Ca2+ uptake, i.e., by activation of voltage-dependent Ca2+ channels in the plasma membrane. In addition, it appears that calmodulin is involved in the process by which glucose and veratridine act on stored calcium to raise the cytosolic Ca2+ concentration. Finally, trifluoperazine did not inhibit insulin release mediated by cyclic AMP (3-isobutyl-1-methyl-xanthine). Two conclusions may be drawn from this finding: (1) calmodulin may not be involved in the process of exocytosis per se and (2) cyclic AMP and glucose seem to mobilize stored calcium by different mechanisms.