The insulinogenic response to a standard i.v. dose of a sulfonylurea can be markedly augmented in normal, trained, conscious dogs if they are given 30 min earlier a single i.v. dose of dihydroergotamine (DHE). Since the parent substance ergotamine possessed no such amplifying properties, further experiments were conducted to clarify the essential structural requirements that have to be fulfilled for an ergot alkaloid to act as an amplifier of sulfonylurea-stimulated insulin secretion. Amine alkaloids ergonovine, dihydroergonovine and dihydromethylergonovine had no amplifying potency, but the hydrogenated amino acid alkaloids dihydroergocornine, dihydroergocristine and dihydroergokryptine (Hydergine) were almost as potent amplifiers as was DHE. The data indicate that (a) DHE, Hydergine and by inference all hydrogenated amino acid alkaloids are potent amplifiers of sulfonylurea-stimulated insulin secretion; (b) saturation of the double bond at C9 and C10 of the lysergic acid moiety and the presence of an amino acid side chain are essential structural requirements for an ergot alkaloid to function as an amplifier of the action of sulfonylureas; and (c) it appears that these compounds are acting chiefly by mechanisms other than α-adrenergic and serotonergic receptor blockade, perhaps as regulatory molecules inducing positive cooperative changes in integral proteins of the plasma membrane of beta cells