The direct effects of cortisol and cortisone on insulin and glucagon secretion by the isolated perfused rat pancreas were examined. Cortisone, but not cortisol, in a final concentration of 10(-4) M acutely and strongly inhibited both phases of glucose- and arginine-induced insulin secretion. In repeated pulse administrations, the second infusion of cortisone elicited an inhibition of insulin release equal to the first response. Glucagon output was slightly suppressed by cortisol and more significantly by cortisone in a glucose-free medium. The blockade of alpha-adrenergic receptors by phentolamine attenuated, but did not prevent, the strong inhibitory action of cortisone on insulin secretion.