We examined the biotransformation of benzothiazole derivatives (BTHs) by an axenic microbial culture. A Gram-negative bacterium, tentatively named as strain HKT554 and identified as Pseudomonas putida, was able to transform not only benzothiazole and 2-mercaptobenzothiazole but also 2-methylthiobenzothiazole, which was previously reported as the dead-end product of wastewater treatment. GC/MS analysis of the solid-phase extract of the culture broth showed the formation of 2-(3H)-benzothiazolone/2-hydroxybenzothiazole from benzothiazole. By transposon mutagenesis, a mutant library containing ca. 5000 insertion mutants was constructed from the P. putida strain HKT554. Analysis of the disrupted gene from one of the mutants showing BTHs transformation deficiency revealed that the knocked-out gene was naphthalene dioxygenase. To our knowledge, this is the first report on the biotransformation of BTHs by Gram-negative bacteria.