Transport of glucose-6-P in intact rat liver microsomes is inhibited by 4,4'-diisothiocyano-1,2-diphenyl[3H]ethane-2,2'-disulfonic acid ([3H]H2DIDS). The concentration of [3H]H2DIDS that inhibits transport activity by 50% is 35 microM. Glucose-6-P protected against the inhibition of transport activity caused by [3H]H2DIDS; mannose-6-P, 2-deoxyglucose-6-P, galactose-6-P, fructose-6-P, or glycerol-2-P did not. [3H]H2DIDS-treated microsomes were solubilized in sodium dodecyl sulfate and the microsomal polypeptides were separated by polyacrylamide gel electrophoresis. Labeled polypeptides were identified by autoradiography. Treatment of microsomes with concentrations of [3H]H2DIDS (50-100 microM) that resulted in maximal inhibition of transport activity allowed the identification of two microsomal polypeptides that contained most of the incorporated radioactivity. Their molecular weights were 54,000 and 59,000. The labeling of the former, but not the latter polypeptide was saturable and correlated linearly with the level of inhibition of transport activity. Concomitantly with its protective effect on translocase activity, the presence of glucose-6-P during the reaction with [3H]H2DIDS resulted in a significant increase in the amount of label incorporated into this peptide. This stimulation of labeling was specific for glucose-6-P. These results implicate the 54,000-dalton polypeptide as an obligatory component of the rat liver microsomal glucose-6-P translocase.