Department of Microbiology, Bose Institute, Kolkata 700054, India Correspondence Tapan K. Dutta tapan{at}bic.boseinst.ernet.in Staphylococcus sp. strain PN/Y, capable of utilizing phenanthrene as a sole source of carbon and energy, was isolated from petroleum-contaminated soil. In the degradation of phenanthrene by strain PN/Y, various metabolites, isolated and identified by a combination of chromatographic and spectrometric analyses, revealed a novel phenanthrene assimilation pathway involving 2-hydroxy-1-naphthoic acid. Metabolism of phenanthrene was initiated by the dioxygenation on the 1,2-position of phenanthrene followed by meta -cleavage of phenanthrene-1,2-diol, leading to 2-hydroxy-1-naphthoic acid, which was then processed via a novel meta -cleavage pathway, leading to the formation of trans -2,3-dioxo-5-(2'-hydroxyphenyl)-pent-4-enoic acid and subsequently to salicylic acid. In the lower pathway, salicylic acid was transformed to catechol, which was then metabolized by catechol-2,3-dioxygenase to 2-hydroxymuconaldehyde acid, ultimately forming TCA cycle intermediates. The catabolic genes involved in phenanthrene degradation were found to be plasmid-encoded. This detailed study of polycyclic aromatic hydrocarbon (PAH) metabolism by a Gram-positive species involving a unique ring-cleavage dioxygenase in a novel phenanthrene degradation pathway provides a new insight into the microbial degradation of PAHs. Abbreviations: d 6 -DMSO, deuterated DMSO; ESI, electrospray ionization; PAH, polycyclicaromatic hydrocarbon The GenBank/EMBL/DDBJ accession number for the sequence determined in this study is DQ404012.