Abstract—Wild-type cells of Rhodococcus pyridinivorans 5Ар were found to be highly efficient naphthalene degraders, completely utilizing this compound (500 mg/L) after 3 days, and may be used for remediation of naphthalene-contaminated aquatic ecosystems. Inactivation of the biodegradation genes narAa (encoding the large subunit of naphthalene dioxygenase) and narB (encoding cis-naphthalene dihydrodiol dehydrogenase) resulted it the loss of ability to use naphthalene as the sole energy source, which indicated the absence in the genome of R. pyridinivorans 5Ар of the determinants responsible for alternative pathways of naphthalene oxidation. Moreover, narB inactivation resulted in accumulation of a polar colored compound (probably a product of primary naphthalene oxidation) in the medium.