Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of > 1% (v/v) toluene in the culture medium. A set of multidrug efflux pumps have been found to play a major role in the tolerance of this bacterium to organic solvents (Rojas et al., J Bacteriol 183: 3967–3973). In the course of studies of the mechanisms underlying solvent tolerance in DOT-T1E, we isolated a spontaneous solvent-sensitive mutant derivative which had lost the genes encoding the TtgGHI efflux pump, the most important extrusion element in quantitative terms. Genomic comparisons between the mutant and its parental strain by microarray analysis revealed that in addition to the ttgVW-ttgGHI gene cluster, another group of genes, highly similar to those found in the Tn4653A and ISPpu12 transposable elements of the TOL plasmid pWW0 from P. putida mt-2, were also absent from this strain. Further analysis demonstrated that strain DOT-T1E harboured a large plasmid (named pGRT1) that was lost from the solvent-sensitive mutant. Mapping analysis revealed that the ttgVW-ttgGHI genes and the Tn4653A-like transposon are borne by the pGRT1 plasmid. Plasmid pGRT1 is highly stable and its frequency of loss is below 10−8 per cell per generation under a variety of growth conditions, including nutritional and physical stresses. The pGRT1 plasmid is self-transmissible, and its acquisition by the toluene-sensitive P. putida KT2440 and Pseudomonas aeruginosa PAO1 increased the recipient's tolerance to toluene up to levels similar to those exhibited by P. putida DOT-T1E. We discuss the importance and potential benefits of this plasmid for the development of bacteria with enhanced solvent tolerance, and its potential impact for bioremediation and whole-cell biotransformations.