The mechanisms by which diazoxide and D600 affect insulin release have been compared in experiments using isolated rat islets. Diazoxide (20–400 (JLM) and D600 (1–50 μM) produced a dose-dependent inhibition of glucose-stimulated release. Diazoxide also inhibited the insulinotropic effect of leucine and related substances (ketoisocaproate and BCH), but not that of potassium or of arginine and other cationic amino acids. Diazoxide suppressed glucose and leucine stimulation of Ca uptake in islet cells, but had no effect on the stimulation by potassium and arginine. By contrast, D600 suppressed the effect of all these agents on both Ca uptake and insulin release. Theophylline partially antagonized the inhibitory effect of D600 on release, in the presence of diazoxide, theophylline was much less effective, except when combined with cationic amino acids. Diazoxide inhibition of glucose-induced release was prevented by phentolamine, but hot by dihydroergotamine and yohimbine, two other blpckers of a-adrenergic receptors. Epinephrine abolished the insulinotropic effect of arginine alone or with theophylline. Diazoxide increased 86Rb+ efflux from islet cells, whereas D600 and epinephrine decreased it. The acceleration of efflux by diazoxide was inhibited by D600 and phentolamine, but not by epinephrine or dihydroergotamine. It thus appears that the effects of diazoxide on B-cells are not due to activation of α-adrenergic receptors. The results suggest that, in contrast to the direct blockade of Ca channels by D600, the blockade of these channels by diazoxide is secondary to the hyperpolarization of the B-cell membrane. Since the latter results from an increase in K permeability, the inhibitory effects of diazoxide are restricted to stimulators that depolarize the B-cell membrane by decreasing its K permeability (glucose, leucine, and related substances) and do not affect the stimulation by K and cationic amino acids, which depolarize by other mechanisms.