The glucose transporter (IIBCGlc/IIAGlc complex) of the bacterial phosphotransferase system couples vectorial translocation to phosphorylation of the transported sugar. The IIAGlc subunit transfers the phosphoryl group from the phosphoryl carrier protein P-HPr to the IIBCGlc subunit. IIBCGlc translocates and phosphorylates glucose. The site of IIBCGlc phosphorylation is cysteine 421 as shown by mass spectrometric and biochemical analyses of phosphorylated peptides. Site-directed mutagenesis of Cys421 (C421S) afforded a stable but completely inactive protein (Nuoffer, C., Zanolari, B., and Erni, B. (1988) J. Biol. Chem. 263, 6647-6655). Cys421 is located in the C-terminal cytoplasmic domain of the IIBCGlc subunit in a sequence context (LDACITRL) which is well conserved in other transporters of the bacterial phosphotransferase system. Phosphocysteine has been shown previously to be the catalytic intermediate of the mannitol transporter (Pas, H. H., Meyer, G. H., Kruizinga, W. H., Tamminga, K. S., van Weeghel, R. P., and Robillard, G. T.