The significance of Ca++ for glucose stimulation of insulin release was studied in an in vitro system with beta-cell-rich pancreatic islets microdissected from oh/ob-mice. There was only a slight depression of cAMP in islets exposed to the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine after withdrawal of Ca++ from the incubation medium. The lack of a stimulatory effect of glucose noted in the absence of extracellular Ca++ is therefore probably accounted for by factors other than impaired adenylate cyclase activity. A rise of extracellular Ca++ above the concentration necessary for obtaining the optimal secretagogic effect of glucose resulted in inhibition of the glucose-stimulated insulin release, leaving basal secretions and islet contents of cAMP unaffected. Evidence was provided in support of the idea that H+ completes for Ca++ in glucose stimulation of insulin release. Both the rate of basal insulin release and that seen after stimulation with glucose were diminished by about 50% after introducing 0.2 mM La+++ in the incubation medium. These observations emphasize the significant role of Ca++ in the regulation of insulin secretion, suggesting that not only a decrease but also an increase of the functionally important intracellular pool(s) of Ca++ can result in a diminished response to glucose.