The bacterial tetracycline/H+ antiporter (TetA) mediates active efflux of a chelation complex of tetracycline with a divalent cation such as Mg2+, Co2+, or Mn2+ [Yamaguchi, A., Udagawa, T., & Sawai, T. (1990a) J. Biol. Chem. 265, 4809-4813]. The positive charge of Arg70 in the antiporter is important for the transport function [Yamaguchi, A., Someya, Y., & Sawai, T. (1992c) J. Biol. Chem. 267, 19155-19162]. Out of six site-directed mutants of Arg70, only the Lys70 mutant retained moderate transport activity, whereas the Ser70, Ala70, Trp70, Leu70, and Asp70 mutants had no or extremely low transport activity. In this study, we constructed the Cys70 mutant and found that the Cys70 mutant showed, unexpectedly, a significant activity comparable to that of the Lys70 mutant in the presence of Co2+ ions, whereas it showed very low activity as well as the Ala70 mutant in the presence of Mg2+ or Mn2+ ions. Hg2+, which is known to be a cysteine specific modifier but has no ability to form a complex with tetracycline, caused a dramatic increase in the Vmax value of Co(2+)-dependent tetracycline transport mediated by the Cys70 mutant without affecting the k(m) value, whereas activities of the wild-type and the Lys70 and Ala70 mutants were not affected by Hg2+, Hg2+ alone without Co2+ could not support the transport activity at all, because Hg2+ does not form a chelation complex with tetracycline. These observations suggest that a mercaptide formed between the SH group of Cys70 and Hg2+ or Co2+ works as a positively charged side chain like that of Arg or Lys. When the SH group of the Cys70 mutant was masked with modification by sulfhydryl reagents, the residual activity was no longer affected by Hg2+. Inversely, when the Cys70 mutant was preincubated with Hg2+, it was protected from the inactivation by sulfhydryl reagents. These observations also confirm the mercaptide formation between the Cys70 and a divalent cation as a functional side chain.