The identity of the microorganisms capable of anaerobic p-xylene degradation under denitrifying conditions is hitherto unknown. Here, we report highly enriched cultures of freshwater denitrifying bacteria that grow anaerobically with p-xylene as the sole organic carbon source and electron donor. Long curved rods, with 95% 16S rRNA gene sequence identity to Denitratisoma oestradiolicum, dominated the enrichment cultures (>91% of all cells), as detected by phylotype-specific probes. These Rhodocyclaceae microorganisms were distantly related to other denitrifying hydrocarbon-degrading Betaproteobacteria from the Azoarcus-Thauera clade. Complete oxidation p-xylene to CO(2) coupled to denitrification was suggested by quantitative measurements of substrate consumption. Metabolite analysis identified (4-methylbenzyl)succinate and (4-methylphenyl)itaconate, suggesting addition to fumarate as an initial activation reaction.