Unique hot spots for sulfhydryl inactivation of Cys mutants of the metal-tetracycline/H⁺ antiporter (TetA) were found at the fourth positions of the dual conserved sequence motifs, GKXXDRXGRR and GRXXXKXGEK [Yamaguchi, A., Kimura, T., Someya, Y., & Sawai, T. (1993) J. Biol. Chem. 268, 6496-6504]. The fourth positions of these motifs are occupied by Ser65 and Ala269, respectively. N-Ethylmaleimide (NEM) rapidly bound to and inactivated the S65C and A269C mutants. M64C and I268C showed low reactivity to NEM, probably due to the partial cripticity of these residues. In contrast, NEM rapidly bound to T270C but did not inactivate it. NEM completely inactivated S65C, whereas a small sulfhydryl reagent, methyl methanethiosulfonate (MMTS), caused only 40% inactivation. [¹⁴C] NEM binding to S65C was inhibited by tetracycline. These observations indicated that position 65 is located close to the substrate-protein interaction site and that the inactivation by sulfhydryl reagents comprises volume-dependent steric hindrance. The S65M mutant, having a side chain analogous to one of the thiomethyl cysteines of MMTS-modified S65C, showed about 30% of the V_max value for the wild-type tetracycline transport, while the S65F mutant had completely lost the activity, confirming the idea of volume-dependent steric hindrance at position 65. On the other hand, the A269C mutant was greatly inactivated by both NEM and MMTS. The degree of the inactivation by MMTS (90%) was higher than that of NEM (80%). The synergetic inactivation observed for the S65C/A269C double mutant suggested different inactivation mechanisms for A269C and S65C. [₁₄C] NEM binding to A269C was not affected by tetracycline, thus, position 269 is far from the tetracycline binding site. The V_max value for the tetracycline transport by the A269M mutant was about 20% of that for the wild-type, while that of the A269F mutant was about 60% of that for the wild type. These observations indicated that the effect of a substituent at position 269 is not directly related to its volume and thus should be a remote conformational one.