A MAJOR mechanism by which insulin stimulates glucose transport in muscle and fat is the translocation of glucose transporters from an intracellular membrane pool to the cell surface1-5. The existence of a distinct insulin-regulatable glucose transporter was suggested by the poor cross-reactivity between antibodies specific for either the HepG2 or rat brain glucose transporters and the rat adipocyte glucose transporter6,7. More direct evidence was provided by the production of a monoclonal antibody (mAb 1F8) specific for the rat adipocyte glucose transporter that immunolabels a species of relative molecular mass 43,000 (43K) present only in tissues that exhibit insulin-dependent glucose transport8, suggesting that this protein may be encoded by a different gene from the previously described mammalian glucose transporters9-13. This antibody has been used to immunoprecipitate a 43K protein that was photoaffinity-labelled with cytochalasin B in a glucose displaceable way, and to immunolabel a protein in the plasma membrane of rat adipocytes, whose concentration was increased at least fivefold after cellular insulin exposure. Here we describe the cloning and sequencing of cDNAs isolated from both rat adipocyte and heart libraries that encode a protein recognized by mAb 1F8, and which has 65% sequence identity to the human HepG2 glucose transporter9. This cDNA hybridizes to an mRNA present only in skeletal muscle, heart and adipose tissue. Our data indicate that this cDNA encodes a membrane protein with the characteristics of the translocatable glucose transporter expressed in insulin-responsive tissues.