Glucose-induced inhibition of Ca⁺⁺ extrusion from the β-cell may contribute to the rise in cytosol Ca⁺⁺ that leads to insulin release. To study whether interference with Na/Ca exchange is involved in this inhibition the effects of glucose were compared to those of ouabain. This substance inhibits Na/K ATPase, decreases the transmembrane Na⁺ gradient in islets, and thus interferes with Na/Ca exchange. Collagenase isolated rat islets were maintained for 2 d in tissue culture with a trace amount of ⁴⁵Ca⁺⁺. Insulin release and ⁴⁵Ca⁺⁺ efflux were then measured during perifusion. In Ca⁺⁺-deprived medium (to avoid changes in tissue specific radioactivity) 16.7 mM glucose inhibited ⁴⁵Ca⁺⁺ efflux. Initially 1 mM ouabain inhibited ⁴⁵Ca⁺⁺ efflux in a similar fashion, the onset being even faster than that of glucose. The effects of 16.7 mM glucose and ouabain were not additive, indicating that both substances may interfere with Na/Ca exchange. In the presence of Ca⁺⁺, 16.7 mM glucose induced biphasic insulin release. Ouabain alone caused a gradual increase of insulin release. Again, the effects of ouabain and 16.7 mM glucose were not additive. In contrast, at a submaximal glucose concentration (7 mM) ouabain enhanced both phases of release. An important role for Na/Ca exchange is suggested from experiments in which Ca⁺⁺ was removed at the time of glucose-stimulation (16.7 mM). The resulting marked inhibition of insulin release was completely overcome during first phase by ouabain added at the time of Ca⁺⁺ removal; second phase was restored to 60%. This could be due to the rapid inhibitory action of ouabain on Ca⁺⁺ efflux thereby preventing loss of cellular calcium critical for glucose to induce insulin release. It appears, therefore, that interference with Na/Ca exchange is an important event in the stimulation of insulin release by glucose.