(Ca2+ + Mg2+)-ATPase enzyme activity of a purified plasma membrane preparation from a glucose responsive rat insulinoma, was characterized as Ca2(+)-dependent dephosphorylation of [gamma-32P]ATP. A high-affinity enzyme with a Km(ATP) ranging from 20 to 30 microM and a Km(Ca2+) of 1 microM was identified. Glucose inhibited this high-affinity enzyme in a dose-dependent manner, with no significant inhibition at a concentration between 0 and 5 mM, 50% inhibition at 13.3 mM and 94.5% inhibition at 30 mM. The inhibitory effect of glucose was immediate and rapidly reversible. The effect was stereospecific for the alpha-anomer. These findings support the concept that glucose acts directly at the beta-cell plasma membrane and is involved in the maintenance of elevated intracellular free calcium concentrations associated with insulin release by directly or indirectly inhibiting energy-dependent calcium efflux. Glyceraldehyde (20 mM) increased enzyme activity 3-fold, while other metabolic fuels had no effect. This suggests that inhibition of the enzyme is not an obligatory requirement for insulin release. Calmodulin stimulated the enzyme activity in calmodulin-depleted but not in undepleted membranes. Trifluoperazine (30-100 microM) inhibited (Ca2+ + Mg2+)-ATPase in a dose-dependent manner (14-61% activity) and the activity was also inhibited by vanadate (0.1-1.0 mM) and NaCl (150 mM).