The primary objective of this study was to evaluate the impact of substrate interactions on the biotransformation rates and mineralization potentials of gasoline monoaromatics and methyltert-butyl ether (MTBE), compounds that commonly co-exist in groundwater contaminant plumes. A mixed culture was derived from gasoline-contaminated aquifer material using toluene as the enrichment substrate. Two pure cultures,Rhodococcussp. RR1 and RR2, were isolated from the mixed culture. The three toluene-grown cultures were shown to biotransform all of the six BTEX compounds (benzene, toluene, ethylbenzene,o-xylene,m-xylene, andp-xylene), both individually and in mixtures, over a broad range of concentrations. The mixed culture was shown to degrade all of the BTEX compounds to14CO2, while the two isolates mineralized BTE(m-/p-)X, but biotransformedo-xylene without production of carbon dioxide. Studies to evaluate substrate interactions caused by the concurrent presence of multiple BTEX compounds during their biodegradation revealed a number of patterns,including competitive inhibition and cometabolism. Ethylbenzene was shown to significantly inhibit BTX degradation in mixtures. MTBE was not biodegraded by any of the three toluene-grown cultures over a range of MTBE concentrations. Furthermore, the presence of MTBE at concentrations of 2 to 100 mg/L had no effect on BTEX biotransformation rates.