Two transposons, Tn4351 and Tn4400, which were originally isolated from the obligate anaerobe Bacteroides fragilis, carry a tetracycline resistance (Tcr) gene that confers resistance only on aerobically grown Escherichia coli. This aerobic Tcr gene, designated tetX, has been shown previously to act by chemically modifying tetracycline in a reaction that appears to require oxygen. We have now obtained the DNA sequence of tetX and 0.6 kb of its upstream region from Tn4400. Analysis of the DNA sequence of tetX revealed that this gene encoded a 43.7-kDa protein. The deduced amino acid sequence of the amino terminus of the protein had homology with a number of enzymes, all of which had in common a requirement for NAD(P). In an earlier study, we had observed that disrupted cells, unlike intact cells, could not carry out the alteration of tetracycline. We have now shown that if NADPH (1 mM) is added to the disrupted cell preparation, alteration of tetracycline occurs. Thus, TetX appears to be an NADP-requiring oxidoreductase. Tn4400 conferred a fivefold-lower level of tetracycline resistance than Tn4351. This finding appears to be due to a lower level of expression of the tetX on Tn4400, because the activity of a tetX-lacZ fusion from Tn4400 was 10-fold lower than that of the same fusion from Tn4351. A comparison of the sequence of the tetX region on Tn4351 with that on Tn4400 showed that the only difference between the upstream regions of the two transposons was a 4-base change 350 bp upstream of the start of the tetX coding region. The 4-base change difference creates a good consensus -35 region on Tn4351 that is not present on Tn4400 and could be creating an extra promoter.