To determine the membrane topology of Tet(K), conventional antibiotic resistance genes were used as reporter molecules. A series of fusion genes comprising a resistance gene-beta-lactamase (amp) or chloramphenicol acetyl transferase (cat)-and one loop of the 14 putative transmembrane segments of Tet(K) was constructed. Escherichia coli TG1 with the tet(K)-amp fusion gene at the site of the putative periplasmic loop showed resistance to ampicillin, but the bacterium with the tet(K)-cat fusion gene at the site of the putative cytoplasmic loop showed resistance to chloramphenicol. These findings supported a topology of 14 membrane-spanning segments for Tet(K). Three exceptional cases were observed, which were apparently due to the presence of an acidic residue, Glu, in the preceding transmembrane segment. Mutants in which these acidic residues was substituted for alanine were also constructed, and the effect of glutamic acid in the transmembrane segment on the topology of the fusion proteins was examined.