We have investigated glucose transport proteins in isolated human adipocytes. Using the cytochalasin B binding assay to measure glucose transporters in subcellular membrane subfractions, we found that insulin induced translocation of intracellular glucose transporters to the cell surface. Isoelectric focusing of glucose transporters photolabeled with [3H]cytochalasin B revealed two distinct glucose transporter isoforms in low density microsomes focusing at pH 5.6 and pH 6.4, but only the pH 5.6 isoform was detectable in plasma membranes and only the pH 6.4 form was found in the high density microsomes. Insulin recruited only the pH 5.6 glucose transporter from the low density microsomes to the plasma membrane with no effect on the pH 6.4 transporter isoform. The results suggest that the pH 6.4 species is an immature form of the glucose transporter initially located in the high-density microsome fraction, which then migrates to the low-density microsomes where it matures (converted to pH 5.6 species) and becomes available for insulin-mediated recruitment to the plasma membrane.