Elevations in intracellular Ca2+ in electrically permeabilized islets of Langerhans produced rapid insulin secretory responses from β-cells, but the Ca2+-induced secretion was not maintained and was irrespective of the pattern of administration of elevated Ca2+. Ca2+-insensitive β-cells responded normally to activators of protein kinase C or cAMP-dependent kinase with increased insulin secretion. The loss of secretory responsiveness to Ca2+ was paralleled by a reduction in Ca2+-induced protein phosphorylation. This was caused by a reduction in Ca2+/calmodulin-dependent protein kinase II (CaMK II) activity in the desensitized cells, as assessed by measuring the phosphorylation of a CaMK II-specific exogenous substrate, autocamtide-2. The Ca2+-induced reductions in kinase activity and protein phosphorylation were not dependent on the activation of Ca2+-dependent protein kinases and were not caused by the activation of phosphoprotein phosphatases or of Ca2+-activated proteases. The concomitant reductions in CaMK II activity and Ca2+-induced insulin secretion suggest that the activation of CaMK II is required for normal insulin secretory responses to increased intracellular Ca2+ concentrations.