The genes for a two-component regulatory system of the denitrifying toluene-degrading bacterium Thauera aromatica were identified immediately upstream of the genes for benzylsuccinate synthase (bssDCAB), the first enzyme involved in anaerobic toluene metabolism. The genes apparently encode the regulators of toluene catabolic enzymes and were therefore termed tdiSR (for toluene degradation inducing sensor and regulator). The tdiR gene product was overproduced in Escherichia coli and assayed for binding to a DNA fragment containing the 5′ region of the bss operon. We observed specific DNA binding with cell extracts containing overproduced TdiR, but not with control extracts. The tdiSR genes are almost identical to two genes of Thauera strain T1, which have not been assigned a function so far. In addition, the derived gene products share similarity with regulators of toluene and styrene catabolic pathways in aerobic Pseudomonas species, and with the tutCB gene products of Thauera strain T1. The latter have previously been implicated in regulating anaerobic toluene metabolism. Our data suggest that toluene catabolism under aerobic and anaerobic conditions is regulated by similar, but distinct two-component systems.