The tbu regulon of Ralstonia pickettii PKO1 encodes enzymes involved in the catabolism of toluene, benzene, and related alkylaromatic hydrocarbons. The first operon in this regulon contains genes that encode the tbu pathway's initial catabolic enzyme, toluene-3-monooxygenase, as well as TbuT, the NtrC-like transcriptional activator for the entire regulon. It has been previously shown that the organization of tbuT, which is located immediately downstream of tbuA1UBVA2C, and the associated promoter (PtbuA1) is unique in that it results in a cascade type of up-regulation of tbuT in response to a variety of effector compounds. In our efforts to further characterize this unusual mode of gene regulation, we discovered another open reading frame, encoded on the strand opposite that of tbuT, 63 bp downstream of the tbuT stop codon. The 1,374-bp open reading frame, encoding a 458-amino-acid peptide, was designated tbuX. The predicted amino acid sequence of TbuX exhibited significant similarity to several putative outer membrane proteins from aromatic hydrocarbon-degrading bacteria, as well as to FadL, an outer membrane protein needed for uptake of long-chain fatty acids in Escherichia coli. Based on sequence analysis, transcriptional and expression studies, and deletion analysis, TbuX seems to play an important role in the catabolism of toluene in R. pickettii PKO1. In addition, the expression of tbuX appears to be regulated in a manner such that low levels of TbuX are always present within the cell, whereas upon toluene exposure these levels dramatically increase, even more than those of toluene-3-monooxygenase. This expression pattern may relate to the possible role of TbuX as a facilitator of toluene entry into the cell.