The irreversible inhibition of glucose transport by 1-fluoro-2,4-dinitrobenzene (FDNB) has been used to identify membrane proteins possibly associated with glucose transport in human erythrocytes. D-Glucose was shown to enhance significantly the rate of FDNB inhibition of transport when present during the reaction, whereas cytochalasin B (CB) and D-maltose retarded this FDNB inhibition of transport. This modulation of the inhibition reaction formed basis for a double isotopic differential labeling technique using [14C]- and [3H] FDNB followed by SDS-polyacrylamide gel electrophoresis to distinguish transport-associated polypeptides from bulk membrane dinitrophenylated proteins. Reactions in the presence of CB or maltose revealed the presence of a differentially labeled polypeptide(s), with a molecular weight of approximately 60,000-65,000 daltons. This effect could in part be reversed in the presence of D-glucose but not L-glucose. Reactions in the presence of D-glucose resulted in two regions of differential labeling. One region was around 200,000 daltons and the other corresponded to a 90,000-dalton band. Extraction of membrane proteins with p-chloromercuribenzene sulfonate resulted in no loss of the 60,000-dalton peak, indicating that this labeled polypeptide(s) was firmly anchored in the hydrophobic core of the membrane. These results indicate that as many as three membrane polypeptides are differentially labeled by FDNB under conditions strongly associated with the inhibition of the glucose transport system and may be involved in the regulation of glucose transport.