The role of both the plasmid pGRT1 and the solvent extrusion pump ttgGHI during toluene biodegradation in Pseudomonas putida DOT-T1E cultures was investigated in a sterile suspended growth bioreactor operated as chemostat and inoculated independently with a wild type strain, a mutant lacking the pGRT1 plasmid (P. putida DOT-TIE-100), and a mutant with a modified pGRT1 plasmid lacking the genes encoding the ttgGHI solvent efflux pump (P. putida DOT-TIE-28). A similar process performance was recorded in all tested strains at 4 g tol m−3 and dilution rates (D) of 0.1 h−1. However, operation at 10 g tol m−3 and D of 0.2 h−1 revealed a much lower toluene EC (285 g m−3 h−1) in P. putida DOT-T1E-100 cultures when compared to wild type and P. putida DOT-T1E-28 cultures (483–498 g m−3 h−1), which suggests that other mechanisms rather than solvent extrusion by the ttgGHI efflux pump supported this superior process performance. Finally, the plasmid pGRT1 analysed exhibited a remarkable stability towards toluene harmful mediated effects, regardless the strain or toluene loading tested.