It has been reported that protein kinase C (PKC) interacts at multiple sites in beta-cell stimulus-secretion coupling. Nevertheless, there is still controversy concerning the importance of this enzyme in glucose-induced insulin release. The present study was undertaken to clarify whether glucose, directly, or through changes in cytoplasmic free Ca2+ concentration, [Ca2+]i, could promote translocation of PKC from the soluble to the membrane compartment. Whereas glucose, which increases [Ca2+]i, did not affect long-term distribution of PKC activity between soluble and membrane fractions, this distribution was reversibly affected acutely by the Ca2+ concentration in the extraction media. Translocation of PKC to the membrane by incubation of HIT cells for 10 min in the presence of 20 nM phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a 5-fold increase in glucose-induced insulin release. This was prevented by 50 nM concentration of the PKC inhibitor staurosporine, provided that the cells were exposed to the inhibitor before the phorbol ester. Cells pretreated with TPA demonstrated increased insulin secretion in response to glucose for several hours. This time course extended beyond the disappearance of [3H]TPA from the cells, which was complete after 1 h. Activation of PKC increased both average insulin release and the amplitude of oscillations 2-fold, but did not affect oscillation frequency. The stimulatory effect of increased PKC activity on insulin release was not matched by changes in [Ca2+]i. We suggest that stimulation of the pancreatic beta-cell with glucose promotes transient translocation of certain PKC isoforms from the cytoplasm to the plasma membrane as a direct consequence of the increase in [Ca2+]i. Such a translocation may promote phosphorylation of one or several proteins involved in the regulation of the beta-cell stimulus-secretion coupling. This results in potentiation of glucose-induced activation of insulin exocytosis, an effect then not mediated by an increase in [Ca2+]i per se. Hence, pulsatile insulin release can be obtained under conditions where overall [Ca2+]i does not change, challenging the view that oscillations in [Ca2+ ]i are indeed driving the oscillations in hormone release.