It has been proposed by others that sulfonylureas exert their extrapancreatic hypoglycemic effects by increasing insulin binding through inhibition of receptor-mediated hormone internalization. In this study, we examined the possibility that the drugs act by inhibiting transglutaminase, an enzyme thought important in the internalization process. For ten days, male rats were fed pulverized chow containing either no drug, glipizide (5 mg/kg initial body wt/d), or tolazamide (75 mg/kg initial body wt/d). Prior to sacrifice, the six-hour fasting level of serum glucose was significantly reduced from 96 mg/100 ml in the control rats to 81 and 42 mg/100 ml in the glipizide- and tolazamide-treated rats, respectively. In contrast, the serum level of insulin was similar for all groups. The activity of transglutaminase in the postnuclear fraction of liver homogenate also was the same for all experimental groups. The specific binding of labeled insulin to purified liver plasma membranes was examined over a broad range of insulin concentrations; once again, there was no difference between experimental groups. Thus, the hypoglycemia caused by sulfonylurea administration could not be attributed to increases in insulin binding, inhibition of transglutaminase activity, or enhanced insulin levels. These data support our previous suggestion, based on in vitro studies, that sulfonylureas act predominately on processes beyond the binding portion of the insulin receptor.