Three strains of non-motile, Gram-positive, filamentous actinomycetes, isolates J4(T), J5 and J59, initially recognized microscopically in activated sludge foam by their distinctive branching patterns, were isolated by micromanipulation. The taxonomic positions of the isolates were determined using a polyphasic approach. Almost-complete 16S rRNA gene sequences of the isolates were aligned with corresponding sequences of representatives of the suborder Corynebacterineae and phylogenetic trees were inferred using three tree-making algorithms. The organisms formed a distinct phyletic line in the Gordonia 16S rRNA gene tree. The three isolates showed 16S rRNA gene sequence similarities within the range 96.9-97.2 % with their nearest phylogenetic neighbours, namely Gordonia bronchialis DSM 43247(T) and Gordonia terrae DSM 43249(T). Strain J4(T) was shown to have a chemotaxonomic profile typical of the genus Gordonia and was readily distinguished from representatives of the genus on the basis of Curie-point pyrolysis mass spectrometric data. The isolates shared nearly identical phenotypic profiles that distinguished them from representatives of the most closely related Gordonia species. It is evident from the genotypic and phenotypic data that the three isolates belong to a novel Gordonia species. The name proposed for this taxon is Gordonia defluvii sp. nov.; the type strain is J4(T) (=DSM 44981(T)=NCIMB 14149(T)).