The biodegradation of nitrobenzene was attempted by using Pseudomonas putida TB 103 which possesses the hybrid pathway combining the tod and the tol pathways. Analysis of the metabolic flux of nitrobenzene through the hybrid pathway indicated that nitrobenzene was initially oxidized to cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene by toluene dioxygenase in the tod pathway and then channeled into the tol pathway, leading to the complete biodegradation of nitrobenzene. A crucial metabolic step redirecting the metabolic flux of nitrobenzene from the tod to the tol pathway was determined from the genetic and biochemical studies on the enzymes involved in the tol pathway. From these results, it was found that toluate-cis-glycol dehydrogenase could convert cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene to catechol in the presence of NAD(+) with liberation of nitrite and the reduced form of NAD(+) (NADH) into the medium.