Elevations in intracellular Ca²⁺ in electrically permeabilized islets of Langerhans produced rapid insulin secretory responses from -cells, but the Ca²⁺-induced secretion was not maintained and was irrespective of the pattern of administration of elevated Ca²⁺. Ca²⁺-insensitive -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.