Classification of strain W-50(T), which was isolated from a wastewater treatment plant, was investigated by a polyphasic approach. Cells of strain W-50(T) were Gram-negative, strictly aerobic, oxidase-positive and yellow-pigmented. Ubiquinone Q-10 was the main respiratory lipoquinone system and polar lipid fingerprints were characterized by the presence of a sphingoglycolipid, suggesting that strain W-50(T) belongs to the alpha-4 subclass of the Proteobacteria. Sequencing and comparative analyses of the 16S rRNA gene of strain W-50(T) supported its chemotaxonomic allocation as an alpha-4 proteobacterium. The most closely related established taxa were species of the genus Sphingopyxis, including Sphingopyxis macrogoltabida (97.3% similarity) and Sphingopyxis terrae (96-4% similarity), and Sphingomonas taejonensis (97.3%). These findings were supported by both the polyamine content, which consisted mainly of spermidine [12.9 micromol (g dry wt)(-1)], and the presence of 2-OH 14:0, 2-OH 15:0 and 2-OH 16:0 in the cellular fatty acid profile. DNA-DNA hybridization experiments resulted in similarity values of 31.9% between strain W-50(T) and Sphingopyxis macrogoltabida IFO 15033(T), 44.9% between strain W-50(T) and Sphingopyxis terrae IFO 15098(T) and 31.0% between strain W-50(T) and Sphingomonas taejonensis KCTC 2884(T). Based upon results obtained by detailed physiological/biochemical testing and previously published molecular evidence, strain W-50(T) was clearly distinguishable from all other Sphingopyxis species. For these reasons, the creation of a novel species, Sphingopyxis witflariensis sp. nov., is proposed; strain W-50(T) (= DSM 14551(T) = CIP 107174(T)) is the type strain.