Pseudomonas putida is well known as a toluene-degrading bacterium. Because methyl tert-butyl ether (MTBE) often coexists with toluene in subsurface environment, in this study we investigated substrate interactions between toluene and MTBE, and their degradation potential in mixtures by Pseudomonas putida. Batch microcosm studies were conducted for a wide concentration range (25–200 mg L−1) to monitor bacterial growth associated with toluene and/or MTBE degradation using Pseudomonas putida previously adapted to toluene. We found that the effect of MTBE on toluene degradation was strongly concentration-dependent, and that concentrations of either toluene or MTBE as low as 25 mg L−1 can enhance degradation of other compound, but those concentrations higher than 25 mg L−1 tend to result in negative or no effect. The negative effect was more pronounced for toluene since MTBE degradation was inhibited at a concentration of 50 mg L−1 or higher toluene. This indicates that concentrations of cocontaminants become a limiting factor for bioremediation of groundwater. Therefore, for higher degradation efficiency, care should be taken in the selection of concentration ranges of toluene and MTBE. © 2010 American Institute of Chemical Engineers Environ Prog, 2011.