Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and NTH derivatives can be detected in diesel oil following hydrodesulfurization treatment, in addition to DBT derivatives. Mycobacterium phlei WU-F1, which possesses high desulfurizing ability toward DBT and its derivatives over a wide temperature range (20-50 degreesC), could also grow at 50degreesC in a medium with NTH or 2-ethylNTH, an alkylated derivative, as the sole source of sulfur. At 50 degreesC, the resting cells of WU-F1 degraded 67% and 83% of 0.81 mM NTH and 2-ethylNTH, respectively, within 8 h. By GC-MS analysis, 2-ethylNTH-desulfurized metabolites were identified as 2-ethylNTH sulfoxide, 1-(2'-hydroxynaphthyl)-1-butene and 1-naphthyl-2-hydroxy-1-butene, and it was concluded that WU-F1 desulfurized 2-ethylNTH through a sulfur-specific degradation pathway with the selective cleavage of carbon-sulfur bonds. Therefore, M. phlei WU-F1 can effectively desulfurize asymmetric organosulfur compounds, NTH and 2-ethylNTH, as well as symmetric DBT derivatives under high-temperature conditions, and it may be a useful desulfurizing biocatalyst possessing a broad substrate specificity toward organosulfur compounds.