Polymyxin B (PMB), a cyclic decapeptide antibiotic, inhibits the hypoglycemic effect of insulin in vivo. To elucidate the mechanism of PMB action, we have studied its effect in vitro on insulin-stimulated pathways in the mouse skeletal muscle. PMB, added to the incubation mixture, specifically inhibited insulin-stimulated 2-deoxyglucose transport and alpha-aminoisobutyric acid uptake in the isolated soleus muscle but did not affect the basal rates of transport (measured in the absence of insulin). PMB did not alter insulin binding and hexokinase activity. PMB effect was observed at all deoxyglucose concentrations tested, and PMB was also able to inhibit vanadate-stimulated glucose transport. By contrast, insulin activation of glycogen synthase was not prevented by PMB. Basal and maximally insulin-stimulated insulin receptor tyrosine kinase activity, tested in a cell-free system, was similar for both autophosphorylation and phosphorylation of exogenous substrates in the absence or in the presence of PMB. Furthermore, the insulin sensitivity of the kinase was increased in the presence of PMB. Our results suggest that the anti-insulin effect of PMB observed in vivo is due to an inhibition of insulin-stimulated glucose transport in the skeletal muscle perhaps through a specific blockade of the insulin-induced translocation of the glucose carriers.