The glucose transporter in the plasma membrane of rat skeletal muscle has been identified by two approaches. In one, the transporter was detected as the polypeptide that was differentially labeled by photolysis with [3H]cytochalasin B in the presence of L- and D-glucose. [3H]Cytochalasin B is a high-affinity ligand for the transporter that is displaced by D-glucose. In the other, the transporter was detected by means of its reaction with rabbit antibodies against the purified glucose transporter from human erythrocytes. By both procedures, the transporter was found to be a polypeptide with a mobility corresponding to a molecular weight of 45,000-50,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis.