Operon OP2 on the Pseudomonas putida TOL plasmid encodes enzymes for m-toluate catabolism; transcription of this operon is activated by XylS in the presence of m-toluate. Because transcriptional activation of OP2 specifically occurs in the stationary phase of growth both in P. putida and in Escherichia coli, we suspected that its transcription is dependent on RpoS (sigmaS). Therefore, we constructed a rpoS disruption strain of P. putida mt-2, and assayed OP2 expression and other phenotypes. OP2 transcription was dependent on rpoS, indicating that the stationary-phase specific activation of OP2 is due to positive regulation by RpoS in P. putida mt-2. The rpoS mutant exhibited reduced viability during the stationary phase and was sensitive to high salt concentrations and H2O2. P. putida mt-2 has two catalase isozymes, KatA and KatB. Expression of the katB gene was specific to the stationary phase and entirely dependent on the rpoS gene, while the katA gene expressed during log phase partially required rpoS. There were no significant changes in tolerance to high temperature or UV light in the rpoS mutant. No difference was observed between the E. coli rpoS mutants and their parents in their capacity to activate OP2, suggesting that the mechanism of OP2 activation in the stationary phase of growth differs between P. putida and E. coli.