1. Islets from normal mice were used to test the acute effects of genistein, a potent tyrosine kinase inhibitor, on stimulus-secretion coupling in pancreatic beta-cells. 2. Genistein produced a concentration-dependent (10-100 microM), reversible, increase of insulin release. This effect was marginal on basal release or in the presence of non-metabolized secretagogues, and much larger in the presence of glucose or other nutrients. The increase in insulin release caused by 100 microM genistein was abolished by adrenaline or omission of extracellular Ca2+. It was not accompanied by any rise of cyclic AMP, inositol phosphate or adenine nucleotide levels. 3. Although genistein slightly inhibited ATP-sensitive K+ channels, as shown by 86Rb efflux and patch-clamp experiments, this effect could not explain the action of the drug on insulin release because the latter persisted when ATP-sensitive K+ channels were all blocked by maximally effective concentrations of glucose and tolbutamide. Genistein was also effective when ATP-sensitive K+ channels were opened by diazoxide and the beta-cell membrane depolarized by 30 mM K, but ineffective in the presence of diazoxide and normal extracellular K. 4. Genistein paradoxically decreased Ca2+ influx in beta-cells, as shown by the inhibition of glucose-induced electrical activity, by the inhibition of Ca2+ currents (perforated patches) and by the lowering of cytosolic [Ca2+]i (fura-2 technique). Genistein thus increases insulin release in spite of a lowering of [Ca2+]i in beta-cells. 5. Daidzein, an analogue of genistein reported not to affect tyrosine kinases, was slightly less potent than genistein on K+ and Ca2+ channels, but increased insulin secretion in a similar way. Three other tyrosine kinase inhibitors, tyrphostin A47, herbimycin A and an analogue of erbstatin variably affected insulin secretion.6. Genistein exerts a number of heretofore unrecognized effects. The unusual mechanisms, by which genistein increases insulin release in spite of a decrease in beta-cell [Ca2+]i and without activating known signalling pathways, do not seem to result from an inhibition of tyrosine kinases.