4-Nitroaniline (4NA), the starting material for the first synthesized azo dye, is a toxic compound found in industrial wastewaters. Several bacterial strains capable of 4NA biodegradation were previously reported but the details of the catabolic pathway were not established. To search for novel metabolic diversity, we isolated a Rhodococcus sp. Strain JS360 by selective enrichment from 4NA-contaminated soil. When grown on 4NA the isolate accumulated biomass released stoichiometric amounts of nitrite and released less than stoichiometric amounts of ammonia, indicating that 4NA was used as sole carbon and nitrogen source to support growth and mineralization. Enzyme assays coupled with respirometry provided preliminary evidence that the first and second steps of 4NA degradation involve monooxygenase-catalyzed reactions followed by ring cleavage prior to deamination. Sequencing and annotation of the whole genome revealed candidate monooxygenases that were subsequently cloned and expressed in E.coli. Heterologously expressed 4NA monooxygenase (NamA) and 4-aminophenol (4AP) monooxygenase (NamB) transformed 4NA to 4AP and 4AP to 4-aminoresorcinol (4AR) respectively. The results revealed a novel pathway for nitroanilines and defined two monooxygenase mechanisms likely to be involved in the biodegradation of similar compounds.