Replacement of extracellular Cl- by isethionate or sulfate during stimulation with glucose or tolbutamide reversibly inhibited insulin release by perifused mouse islets. The concentration of ionized Ca2+ was decreased by 30 and 55% in isethionate and sulfate solutions, respectively. If this fall was prevented, the inhibition of release was only slightly affected (isethionate) or substantially attenuated (sulfate). In conclusion, the inhibition of insulin release occurring in Cl(-)-free solutions cannot be completely ascribed to a decrease in ionized Ca2+ in the medium. The contribution of this latter depends on the Cl- substitute.