Insulin stimulation of glucose transport involves the translocation of vesicles containing the glucose transporter Glut 4 to the plasma membrane. Rab proteins, which have been implicated in the regulation of vesicular traffic, were studied in adipocytes. Rab3B, Rab3C, Rab4, and Rab8 were detected, but Rab3A was not. In the absence of insulin, Rab3B and Rab3C were cytosolic, while Rab4 and Rab8 were associated with membranes. Only Rab4 distribution was modified by insulin. In unstimulated adipocytes, most of Rab4 was found in a low density microsomal fraction, which also contained the majority of Glut 4. After insulin treatment, a 50% decrease in Rab4 content was observed, concomitantly with a departure of transporters to the plasma membrane. The dose responses for the departure of Glut 4 and Rab4 from the microsomal fractions were superimposable, half-maximal effects being obtained with 0.1 nM insulin. Rab4 was redistributed to the cytosol and its movement was reversed by insulin withdrawal. When Glut 4-containing vesicles were immunopurified with antibodies to Glut 4, Rab4 was found in the immune pellets, suggesting that Rab4 was tightly associated with the vesicles. Okadaic acid, an inhibitor of phosphatases 1 and 2A that is known to stimulate Glut 4 translocation, caused the same movement of Rab4 from low density microsomal fraction to the cytosol, while the phorbol ester 12-O-tetradecanoylphorbol-13-acetate had no effect. We suggest that insulin and okadaic acid induce a cycling of Rab4 from a vesicular fraction containing the Glut 4 transporter to the cytosol and that this cycling may participate in the insulin stimulatory action on glucose transporter translocation.