Tryptic digestion studies of the human erythrocyte glucose carrier have shown that a reactive and transport-sensitive exofacial sulfhydryl is located in the carboxy-terminal half of the molecule, corresponding to Cys347, Cys421, or Cys429. In the present studies, the erythrocyte glucose carrier labeled on the exofacial sulfhydryl with bis(maleimidomethyl) ether-L-[35S]cysteine was chemically cleaved, either at tryptophans by N-bromosuccinimide or at nonalkylated cysteines by 2-nitro-5-thiocyanobenzoic acid. The resulting fragments were separated by linear gradient polyacrylamide gel electrophoresis, and the labeled fragments were identified by their apparent molecular weight, and by immunoblotting with antibodies to specific regions of the carrier protein. All of the labeled fragments were recognized by an antibody to the carboxy terminus of the carrier, but not by an antibody to a cytoplasmic loop on the C-terminal half of the carrier. The labeled exofacial sulfhydryl was assigned to Cys429, since this is the only residue of the three possibilities which is beyond the expected cleavage sites of the two reagents in the carrier sequence. These results concur with the predictions of hydropathy analysis and will be relevant for studies of how modification of this sulfhydryl affects carrier function, particularly since several other known carrier isoforms lack a corresponding cysteine.