Accumulation and subcellular localization of dopamine (DA) in pancreatic B-cells and its effects on insulin secretion were investigated in mice following a single injection of L-3,4-dihydroxyphenylalanine (L-DOPA). Electron microscopic autoradiography showed that 3H-DA formed from administered 3H-DOPA was present over B-cells as well as over other types of islet cells. Pretreatment of the animals with a decarboxylase inhibitor greatly reduced the number of autoradiographic grains. In the B-cells the 3H-DA-grains were associated with the secretory granules. The location of the label may suggest an incorporation in the periphery of the beta-granule, rather than in the dense core, supposed to contain insulin. Accumulation of DA in the B-cells following L-DOPA administration was found to inhibit partially the insulin secretory response to different insulin secretagogues (glucose, glibenclamide and L-isopropylnoradrenaline (L-IPNA). Treatment with monoamine oxidase inhibitor + L-DOPA induced an almost total suppression of L-IPNA-stimulated insulin secretion, whereas glucose-induced insulin release was still only partially inhibited. Pretreatment with a decarboxylase inhibitor abolished the effects of L-DOPA. It is suggested that intracellularly accumulated DA in the B-cell exerts an inhibitory action on insulin releasing mechanisms induced by different secretagogues and that this action might involve interference with a calcium translocation process at the level of the secretory granule.