Insulin stimulates glucose transport into adipocytes, at least in part, via the translocation of intracellular transporters to the plasma membrane. The human HepG2-type transporter, which is not insulin-responsive in its native cell type, was expressed in 3T3-L1 adipocytes by infection with recombinant retrovirus harboring the HepG2 transporter cDNA in order to determine whether glucose transporter translocation in adipocytes is restricted to a distinct insulin-sensitive transporter species. The distributions of the endogenous murine and the HepG2 transporters were estimated by quantitative immunoblot analysis of subcellular fractions probed with either a monoclonal antibody that recognized only the human transporter or a polyclonal antibody that recognized both transporter species. In the basal state, the intracellular membrane fraction comprised approximately 50% of the total of each transporter type. Insulin decreased the content of both transporter species in the intracellular membranes by approximately 50% and increased the plasma membrane content of both species by approximately 1.5-2-fold. The similar insulin-mediated increase in the plasma membrane content of endogenous murine and HepG2 glucose transporters was verified by labeling of cell surface glycoproteins with [3H]NaBH4 followed by immunoprecipitation with glucose transporter antibodies. These data indicate that insulin-mediated translocation in 3T3-L1 adipocytes is not restricted to a tissue-specific insulin-responsive glucose transporter species and suggest that other tissue-specific factors regulate the translocation process.