The mannitol permease, an enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) of Escherichia coli, carries out the transport and phosphorylation of D-mannitol in this organism. Previous studies have shown that His-554 and Cys-384 in the mannitol permease are sequentially phosphorylated in reactions necessary for the transport and phosphorylation of the substrate. These residues are located in a large cytoplasmic domain of the protein. Interaction of the permease with mannitol, and its membrane translocation, however, must involve the N-terminal, transmembrane domain (EIIC domain) of the protein. In this report, we use site-directed mutagenesis and mutant complementation to investigate the role of His-195 in the EIIC domain of the mannitol permease, a residue that is conserved in many PTS permeases. In a previous report [Weng, Q.-P., Elder, J., & Jacobson, G. R. (1992) J. Biol. Chem. 267, 19529-19535], we inferred a role for His-195 that involves its hydrogen-bonding ability. Here we show that His-195 plays a role in high-affinity mannitol binding. Moreover, mutant complementation studies show that a functional His-195 must be on the same subunit as a functional Cys-384 in a permease dimer for phosphotransfer to mannitol to occur. These results and kinetic studies of His-195 mutant proteins imply that His-195 also may play an important role in this phosphotransfer reaction. His-195 is predicted to be in a cytoplasmic "loop" in the EIIC domain of the mannitol permease, in which several other residues have been shown to have roles in mannitol permease activity.