The effect of exogenous acid amyloglucosidase on sulphonylurea-induced insulin release was investigated in mice and rats. 1. Pretreatment of mice with acid amyloglucosidase enhanced insulin release induced by the different sulphonylurea derivatives, carbutamide, tolbutamide, glibenclamide, and glibornuride. 2. A dose-response relationship between glibenclamide-induced insulin response and amyloglucosidase dosage covering a 64-fold concentration range was established in mice. 3. Pretreatment of the animals with other macromolecules of similar physiological or chemical properties to acid amyloglucosidase such asα-amylase,β-glucuronidase and albumin did not influence glibenlamide-induced insulin release. 4. The effect of acid amyloglucosidase pretreatment on insulin release induced by different agents known to affect the islet-cell adenylate cyclase-cyclic AMP system such as secretin, L-isopropylnoradrenaline (L-IPNA), arginine, glibenclamide and 3-isobutyl-1-methylxanthine (IBMX) was tested. It was observed that in animals pretreated with acid amyloglucosidase, insulin release was enhanced when stimulated by glibenclamide, a phosphodiesterase inhibitor, but it was similarly enhanced by arginine, a phosphodiesterase activator. Insulin release induced by secretin, L-IPNA, and IBMX was unaffected. 5. Acid amyloglucosidase pretreatment in rats enhanced plasma immunoreactive insulin levels following glibenclamide injection not only in the peripheral veins but also in the portal vein. 6. Mice fasted for 24 hrs displayed a markedly depressed insulin response to tolbutamide injection. Pretreatment of the fasted animals with acid amyloglucosidase could restore the tolbutamide-induced insulin release to the same level as that recorded in a group of freely fed mice. It is suggested that acid amyloglucosidase plays an important role in insulin secretion induced by sulphonylureas. Most evidence suggests that this effect is exerted within the B-cell although an additional effect on the liver cannot be ruled out.