An important signal involved in glucose-stimulated insulin secretion is transduced through the action of a lysosomal acid, glucan 1,4--glucosidase. We investigated the Ca²⁺ dependency of this enzyme activity in relation to insulin release. In isolated islets, increased levels of extracellular Ca²⁺ induced a large increase in acid glucan 1,4--glucosidase activity accompanied by a similar increase in insulin release at both substimulatory and stimulatory concentrations of glucose. At low glucose the Ca²⁺ "inflow" blocker nifedipine unexpectedly stimulated enzyme activity without affecting insulin release. However, nifedipine suppressed ⁴⁵Ca²⁺ outflow from perifused islets at low glucose and at Ca²⁺ deficiency when intracellular Ca²⁺ was mobilized by carbachol. This nifedepine-induced retention of Ca²⁺ was reflected in increased acid glucan 1,4--glucosidase activity. Adding different physiological Ca²⁺ concentrations or nifedipine to islet homogenates did not increase enzyme activity. Neither selective glucan 1,4--glucosidase inhibition nor the ensuing suppression of glucose-induced insulin release was overcome by a maximal Ca²⁺ concentration. Hence, Ca²⁺-induced changes in acid glucan 1,4--glucosidase activity were intimately coupled to similar changes in Ca²⁺-glucose-induced insulin release. Ca²⁺ did not affect the enzyme itself but presumably activated either glucan 1,4--glucosidase-containing organelles or closely interconnected messengers.