Proline binding activity of the Escherichia coli Na+/proline symport carrier is inhibited by a sulfhydryl reagent, N-ethylmaleimide (NEM). Proline and its analogs protected the carrier against the NEM-inactivation in a Na+ (or Li+)-dependent manner. Na+ alone, even in the absence of proline, partially protected it from the NEM-inactivation. Mutant proline carriers, CS281, CS344 and CS349, which have a serine residue in place of Cys-281, Cys-344 and Cys-349, respectively (Yamato, I. and Anraku, Y. (1988) J. Biol. Chem. 263, 16055-16057) were also analyzed for cation-dependent proline binding and NEM-sensitivity. Proline binding activities of CS281 and CS344 were almost completely resistant to NEM, whereas that of CS349 was not. Furthermore, the proline binding activity of CS344 was remarkably lower than those of the wild-type, CS281 and CS349 carriers. These results indicate that Cys-344, which is located in the putative eighth membrane-spanning domain in the carrier, is a cysteine residue functionally involved in the high-affinity binding for sodium ion and proline.