The effects of 5-hydroxytryptamine and 5-hydroxytryptophan on insulin release and 45Ca2+ uptake in islets microdissected from ob/ob mice were studied. At a concentration of 4 mmol/l both compounds slightly stimulated insulin release at a low glucose concentration (3 mmol/l). Insulin release induced by 20 mmol/l D-glucose was inhibited by 4 mmol/l 5-hydroxytryptamine but potentiated by 4 mmol/l L-5-hydroxytryptophan. Mannoheptulose (20 mmol/l) blocked the combined effects of 20 mmol/l D-glucose and 4 mmol/l L-5-hydroxytryptophan on insulin release. 45Ca2+ uptake was inhibited by 4 mmol/l 5-hydroxytryptamine and stimulated by 4 mmol/l L-5-hydroxytryptophan. Mannoheptulose (20 mmol/l) did not affect the 45Ca2+ uptake induced by the latter. When 4 mmol/l L-5-hydroxytryptophan was present only during the 30-min preincubation period. 20 mmol/l-glucose-induced insulin release and 45Ca2+ uptake during a subsequent incubation period were inhibited. Externally added 5-hydroxytryptamine (4 mmol/l) did not change the effects of 4 mmol/l L-5-hydroxytryptophan on insulin release and 45Ca2+ uptake. It is concluded that, when added directly into the incubation medium, 5-hydroxytryptophan has effects on insulin release and 45Ca2+ uptake which are opposite to those observed when 5-hydroxytryptamine is added. These effects do not seem to be mediated by 5-hydroxytryptamine formed intracellularly from 5-hydroxytryptophan.