Increase of the glucose concentration from 3 to 11 mM resulted in a triphasic release of insulin from perifused ob/ob-mouse beta-cells. A slight inhibition was followed after 2 min by a marked peak and a less pronounced sustained response. At the lower glucose concentration glycine had only marginal effects. However, in the presence of 11 mM glucose, 1-10 mM glycine triggered an immediate and dose-dependent response with an initial peak of insulin release followed by sustained stimulation. In individual beta-cells, rise of the glucose concentration from 3 to 11 mM induced initial lowering of the cytoplasmic Ca2+ concentration ([Ca2+]i) followed by large amplitude oscillations from a level of 50-90 nM to peak values exceeding 300 nM. Already at a concentration of 1 mM, glycine transformed the oscillatory pattern into a sustained level with increase of time-average [Ca2+]i. This elevation became more pronounced in the presence of 10 mM glycine. The effects of glycine on insulin release and [Ca2+]i required extracellular Na+ and were reproduced with the N-methyl analogue sarcosine. It is suggested that glycine potentiation of secretion reflects the elevation of time-average [Ca2+]i both by increased entry and reduced elimination of the cation from the cytoplasm.