The solventogenic clostridia have a considerable capacity to ferment carbohydrate substrates with the production of acetone and butanol, making them attractive organisms for the conversion of waste materials to valuable products. In common with other anaerobes, the clostridia show a marked dependence on the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) to accumulate sugars and sugar derivatives. In this study, we demonstrate that extracts of Clostridium beijerinckii grown on N-acetylglucosamine (GlcNAc) exhibit PTS activity for the amino sugar. The PTS encoded by the divergent genes cbe4532 (encoding the IIC and IIB domains) and cbe4533 (encoding a IIA domain) was shown to transport and phosphorylate GlcNAc and also glucose. When the genes were recombined in series under the control of the lac promoter in pUC18 and transformed into a phosphotransferase mutant (nagE) of Escherichia coli lacking GlcNAc PTS activity, the ability to take up and ferment GlcNAc was restored, and extracts of the transformant showed PEP-dependent phosphorylation of GlcNAc. The gene products also complemented an E. coli mutant lacking glucose PTS activity but were unable to complement the same strain for PTS-dependent mannose utilization. Both GlcNAc and glucose induced the expression of cbe4532 and cbe4533 in C. beijerinckii, and consistent with this observation, extracts of cells grown on glucose exhibited PTS activity for GlcNAc, and glucose did not strongly repress utilization of GlcNAc by growing cells. On the basis of the phylogeny and function of the encoded PTS, we propose that the genes cbe4532 and cbe4533 should be designated nagE and nagF, respectively.