Cysteine-scanning mutants, E32C to G62C, of the metal-tetracycline/H؉ antiporter were constructed in order to precisely determine the membrane topology around putative transmembrane segment II. None of the mutants lost the ability to confer tetracycline resistance, indicating that the cysteine mutation in each mutant did not alter the protein conformation. [14 C]N-Ethylmaleimide (NEM) binding to these cysteine mutants in isolated membranes was then investigated. The peptide chain of this region passes through the membrane at least once because residues 36 and 65 are exposed on the outside and inside surfaces of the membrane, respectively (Kimura, T., Ohnuma, M., Sawai, T., and Yamaguchi, A. (1997) J. Biol. Chem. 272, 580 -585). However, there was no continuous segment in which all of the introduced cysteine residues showed almost no reactivity with [14 C]NEM. The proportion of the unbound positions in the second half downstream from position 45 was 55% (10/18), which was clearly higher than that in the first half (21%; 3/14), suggesting that the second half is a transmembrane segment. Positions reactive to NEM appear periodically in the second half. They are located on one side of the helical wheel, suggesting that this side of the transmembrane helix faces a water-filled channel. The cysteine mutants as to the reactive positions in the second half were severely inactivated by NEM except for the P59C mutant, whereas the A40C mutant was the only one inactivated by NEM in the first half. These results suggest that the water-filled channel along this helical region may be a substrate translocation pathway.The tetracycline/H ϩ antiporter (Tet(B)) is a tetracycline exporter existing in the cytoplasmic membrane of Gram-negative bacteria (1-3). This transporter is a member of the major facilitator superfamily (4, 5) including transporters for sugars, amino acids, antibiotics, and neurotransmitters. These transporters were predicted to have 12 transmembrane segments in common (4). The secondary structures of most intrinsic membrane proteins have been determined by the gene fusion method (6, 7). We established a new method for determining the membrane topology on the basis of the reactivity of SH reagents to cysteine-scanning mutants of the tetracycline/H ϩ antiporter ( Fig. 1) (8). Cysteine-scanning mutagenesis of intrinsic membrane proteins, especially lactose permease, has been widely used for site-directed chemical modification analysis (9 -21). We found that Tet(B) has some segments composed of about 20 consecutive residues at which cysteine-scanning mutants were hardly reactive with NEM 1 (22, 23). These segments are embedded in the hydrophobic interior of the membrane. On the basis of this finding, we succeeded in determining the precise boundaries of transmembrane segment (TM) III (23) and TM IX (22). Similar results were obtained for the second transmembrane segment of EmrE.2 However, these observations were inconsistent with the results for lactose permease (10 -16) and UhpT (9), of which some cysteine mutants ...