Excessive endogenous production or exogenous administration of human growth hormone (hGH) causes insulin resistance at both the hepatic and extrahepatic levels. However, which extrahepatic tissues are involved have not been defined. We have examined the diabetogenic action of authentic biosynthetic hGH on whole body glucose disposal, hepatic glucose output, and glucose metabolism in individual peripheral tissues. The use of a highly purified preparation of the hormone allowed us to examine the isolated effects of 22K hGH. The euglycemic hyperinsulinemic (∼ 100 mU/L) clamp plus 3H-2-deoxyglucose technique was used to quantitate the effects of hGH on insulin action in vivo. Administration of biosynthetic hGH at a dose of 10 IU/kg 24 h for 48 hours in male Wistar rats (∼340 g) produced a highly significant decrease in the steady state clamp glucose infusion rate (GIR) when compared with controls (8.1 ± 0.6 v 18.7 ± 0.7 mg/kg/min, P < .001), reduced insulin-mediated suppression of hepatic glucose output (Ra) (3.9 ± 0.6 v 0.7 ± 0.3 mg/kg/min, P < .05) and a decreased clamp glucose disposal rate (Rd) (12.0 ± 0.4 v 18.10 ± 1.1 mg/kg/min, P < .001). There was a significant decrease in insulin-mediated glucose uptake as indicated by tissue accumulation of [3H]-2-deoxyglucose phosphorylation in diaphragm and hindlimb muscles. Insulin action was more substantially reduced in muscles (∼ 50%) than in adipose tissues (∼ 20%). These studies confirm that the diabetogenic action of hGH in the rat is due to a combination of inhibition of insulin suppression of hepatic glucose output and inhibition of the uptake and subsequent utilization of glucose in skeletal muscles.