Rhodococcus sp. PN1 was isolated as a 4-nitrophenol (4-NP) degrading bacterium and can utilize 4-NP as a sole carbon, nitrogen, and energy source. This bacterium also degrades polynitrophenols such as 2,4-dinitrophenol and 2,4,6-trinitrophenol (picric acid). To elucidate the nitrophenol degradation pathways of strain PN1, a genomic library of strain PN1 was constructed using a Rhodococcus host-vector system and the genes encoding the pathways were screened from the library. Consequently, the gene clusters (nphRA1A2 and npdGIJ) encoding 4-NP hydroxylation and picric acid reduction were obtained from the library, respectively. The recombinant Rhodococcus strain containing the nphRA1A2 genes converted 4-NP into 4-nitrocatechol quantitatively, while that containing the npdGIJ genes transformed picric acid into its hydride Meisenheimer complex, quantitatively. Thus, it was found that strain PN1 has two quite different degradation mechanisms for nitrophenols, an oxygenation (4-NP hydroxylation) and a reduction (hydride transfer to the aromatic-ring of picric acid). Sequence analysis of the gene clusters suggested that the former encodes a member of the two component flavin diffusible monooxygenase family (NphA1 and NphA2), while the latter encodes an NADPH-dependent F 42, reductase (NpdG) and an F-420-dependent picric acid reductase (Npd1). Simultaneous degradation of 4-NP and picric acid by strain PN1 indicated that both degradation mechanisms can work simultaneously without significant inhibition. This capability of strain PN1 may contribute to the treatment of industrial wastewaters containing many kinds of nitrophenols.