In isolated adipocytes, polymyxin B inhibited insulin-induced glucose incorporation into lipids in a dose-dependent manner, while polymyxin E, a structurally related antibiotic, was ineffective. To approach the mechanism of this effect, the subcellular distribution of the glucose transporter Glut 4 was investigated. Adipocytes were pretreated without or with polymyxin B before insulin stimulation, subcellular fractionation was performed and Glut 4 was detected by immunodetection. Incubation of adipocytes with polymyxin B prevented the insulin-induced appearance of Glut 4 in the plasma membranes, but did not prevent their decrease from the low-density microsomal fraction. A lower purity of the plasma membrane fractions, a detergent effect of polymyxin B on the membranes or an interference of the substance with the immunodetection of the Glut 4 molecules were excluded. These results suggest that polymyxin B was interfering with the Glut 4 translocation process stimulated by insulin in adipocytes. In a similar fashion, polymyxin B inhibited the insulin-induced increase in IGF II binding to adipocytes. This resulted from a blockade of the appearance of IGF II receptors in the plasma membranes. Since low-molecular-mass GTP-binding proteins have been implicated in the regulation of vesicular trafficking, we have used [alpha-32P]GTP binding to analyze such proteins in adipocyte fractions, after SDS/PAGE and transfer to nitrocellulose. Specific and distinct subsets of GTP-binding proteins were revealed in plasma membrane and low-density microsomal fractions of control adipocytes, whether they were stimulated or not with insulin. Polymyxin B treatment of adipocytes markedly modified the profile of the low-molecular-mass GTP-binding proteins in plasma membranes, but not in low-density microsomal fractions. Our results suggest that polymyxin B was interfering with the exocytotic process of the Glut 4 and IGF II receptor-containing vesicles, perhaps at the fusion step between vesicles and plasma membranes.