The effects of stimulated metabolism on the cytoplasmic Ca2+ concentration (Ca2+i) of insulin-releasing pancreatic beta-cells were studied. When the glucose concentration was increased from 5 to 20 mM, some cell preparations responded with initial lowering of Ca2+i followed by a rise, whereas Ca2+i only increased in others. After prolonged exposure to 5 or 10 mM of the sugar, depolarization with high concentrations of sulfonylurea or K+ caused rapid increases of Ca2+i. However, when subsequently raising glucose to 20 mM there were pronounced temporary decreases of Ca2+i. Marked Ca2+i reducing effects were also obtained after prolonged exposure to 20 mM glucose, when metabolism was augmented further by exposure to leucine or beta-2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid. The results indicate that buffering of Ca2+i is not an artifact but may have physiological significance.