The efflux of radioactivity after loading with trace amounts of tritiated 5-hydroxytryptamine ([ ³ H]5-HT) or 5-hydroxytryptophan ([ ³ H]5-HTP) was studied in perifused β-cellrich pancreatic islets from ob/ob mice. Analysis of the effluent revealed that more than 90% of the radioactivity was released as [ ³ H]5-HT after loading with [ ³ H]5-HTP. Increasing the concentration of glucose in the perifusion medium from 3 to 20 mmol/l enhanced the efflux when islets from fed mice were used and this effect was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Whereas 20 m m -glucose alone did not stimulate the efflux of 5-HT from islets isolated from mice starved for 3 days, a stimulatory effect was observed in the presence of IBMX. Stimulation of the efflux of radioactivity by glucose was inhibited if calcium was omitted from or adrenaline added to the medium. The results are consistent with the concept of exocytotic release of 5-HT occurring in response to stimulation of insulin secretion, although basal non-exocytotic transport must also be occurring across the β-cell membrane.