In Pseudomonas putida DOT-T1E multidrug efflux pumps of the resistance-nodulation-division family make a major contribution to solvent resistance. Two pumps have been identified: TtgABC, expressed constitutively, and TtgDEF, induced by aromatic hydrocarbons. A double mutant lacking both efflux pumps was able to survive a sudden toluene shock if and only if preinduced with small amounts of toluene supplied via the gas phase. In this article we report the identification and characterization in this strain of a third efflux pump, named TtgGHI. The ttgGHI genes form an operon that is expressed constitutively at high levels from a single promoter. In the presence of toluene the operon is expressed at an even higher level from two promoters, the constitutive one and a previously unreported one that is inducible and that partially overlaps the constitutive promoter. By site-directed mutagenesis we constructed a single ttgH mutant which was shown to be unable to survive sudden 0.3% (vol/vol) toluene shocks regardless of the preculture conditions. The mutation was transferred to single and double mutants to construct mutant strains in which two or all three pumps are knocked out. Survival analysis of induced and noninduced cells revealed that the TtgABC and TtgGHI pumps extruded toluene, styrene, m-xylene, ethylbenzene, and propylbenzene, whereas the TtgDEF pump removed only toluene and styrene. The triple mutant was hypersensitive to toluene, as shown by its inability to grow with toluene supplied via the vapor phase.