Polycyclic aromatic hydrocarbons (PAH) are widespread environmental pollutants of considerable risk to human health. The aerobic degradation of PAH via oxygenase reactions has been studied for several decades. In contrast, it was not until very recent that the first key enzyme involved in anaerobic PAH degradation, the dearomatizing 2-naphthoyl-CoA reductase, was isolated and characterized. In this work, a PCR-based functional assay was developed to detect microorganisms that have the ability to anaerobically degrade naphthalene, as a model for larger PAH. The degenerative oligonucleotide probes introduced here amplified a highly conserved region of the gene encoding 2-naphthoyl-CoA reductase (Ncr) in numerous sulfate-reducing pure cultures and environmental enrichments. The assay provides the first molecular tool for monitoring the anaerobic degradation of a model PAH.