Pseudomonas sp. strain HKT554 degrades naphtho[1,2-b]thiophene and two other isomers, naphtho[2,1-b]thiophene and naphtho[2,3-b]thiophene, by cometabolism, in the absence of any specific inducer, at similar degradation rates. A mutant of strain HKT554, deficient in dibenzothiophene degradation, was generated by using a recently developed transposition system. Sequence analysis of the mutant revealed that the knocked out gene was almost identical to naphthalene dioxygenase (EC 1.14.12.12). The mutant, HKT554M, degraded neither the naphthothiophene isomers nor dibenzothiophene, suggesting that the naphthalene dioxygenase is responsible for the initial catabolic reactions onto naphthothiophenes and dibenzothiophene.