Three transmembrane aspartyl residues play essential roles in the transposon Tn10-encoded metal-tetracycline/H+ antiporter (Tet(B)) [Yamaguchi, A. et al. (1992) J. Biol. Chem. 267, 7490-7498]. The tetK gene-encoding tetracycline resistance protein (Tet(K)) of Staphylococcus aureus mediates metal-tetracycline/H+ antiport similarly to Tet(B); however, it has no transmembrane aspartyl residue. On the other hand, Tet(K) has three glutamyl residues, Glu-30, Glu-152 and Glu-397, in the putative transmembrane regions. In the present work, tet(K) gene was expressed in Escherichia coli and the transport activity was measured in everted membrane vesicles. When these glutamyl residues were replaced with Gln, the tetracycline transport activity was almost completely lost, indicating the important roles of these residues in Tet(K). In the case of Glu-397, even the charge-conserved mutation to Asp caused complete loss of the activity. On the other hand, the mutation of Glu-30 and Glu-152 to Asp resulted in significant retention of transport activity. These results are similar to those on the mutation of the three transmembrane aspartyl residues in Tet(B), indicating that the transmembrane glutamyl residues in Tet(K) play roles similar to those of the transmembrane aspartyl residues in Tet(B).