For the complete biodegradation of a mixture of benzene, toluene, and p-xylene (BTX), a critical metabolic step that can connect two existing metabolic pathways of aromatic compounds (the tod and the tol pathways) was determined. Toluate-cis-glycol dehydrogenase in the tot pathway was found to attack benzene-cis-glycol, toluene-cis-glycol, and p-xylene-cis-glycol, which are metabolic intermediates of the tod pathway. Based on this observation, a hybrid strain, Pseudomonas putida TB101, was constructed by introduction of the TOL plasmid pWWO into P. putida F39/D, a derivative of P. putida F1, which is unable to transform cis-glycol compounds to corresponding catechols. The metabolic flux of BTX into the tod pathway was redirected to the tot pathway at the level of cis-glycol compounds by the action of toluate-cis-glycol dehydrogenase in P. putida TB101, resulting in the simultaneous mineralization of BTX mixture without accumulation of any metabolic intermediates. The profile of specific degradation rates showed a similar pattern as that of the specific growth rate of the microorganism, and the maximum specific degradation rates of benzene, toluene, and p-xylene were determined to be about 0.27, 0.86, and 2.89 mg/mg biomass/h, respectively. P. putida TB101 is the first reported microorganism that mineralizes BTX mixture simultaneously.