The purpose of this study was to investigate whether in vivo nitric oxide synthase (NOS) inhibition influences insulin-mediated glucose disposal in rat peripheral tissues. The NOS inhibitorN ^G-nitro-l-arginine methyl ester (l-NAME) or saline was infused constantly during a hyperinsulinemic-euglycemic clamp in normal rats. Glucose utilization rates of insulin-sensitive tissues (individual muscles, heart, and adipose tissues) were simultaneously determined using tracer infusion of 2-deoxy-d-[³H]glucose (2-[³H]DG). NOS blockade with l-NAME resulted in significant (P < 0.05) reduction in both whole body glucose disposal (−16%,P < 0.01) and plasma 2-[³H]DG disappearance rate (−30%, P < 0.05) during hyperinsulinemic-euglycemic clamp.l-NAME significantly decreased insulin-stimulated glucose uptake in heart (−62%,P = 0.01), soleus (−42%, P = 0.05), red (−53%, P < 0.001) and white (−62%, P < 0.001) gastrocnemius, tibialis (−57%,P < 0.01), and quadriceps (−33%, P < 0.05) muscles. The NOS inhibitor also decreased insulin action in brown interscapular (−47%, P < 0.01), retroperitoneal (−52%, P = 0.07), and gonadal (−66%, P = 0.06) adipose tissues. In contrast to in vivo NOS blockade,l-NAME failed to affect basal or insulin-stimulated 2-[³H]DG transport in isolated soleus or extensor digitorum longus muscles in vitro. These results support the hypothesis that the action of insulin to augment glucose uptake by skeletal muscles and other peripheral insulin-sensitive tissues in vivo is NO dependent.