Isolate RS1(T) isolated from used metalworking fluid was found to be a Gram-negative, motile, and non-spore forming rod. Based on phylogenetic analyses with 16S rRNA, isolate RS1(T) was placed into the mendocina sublineage of Pseudomonas. The major whole cell fatty acids were C-18:1 omega 7c (32.6%), C-16:0 (25.5%), and C-15:0 ISO 2OH/C-16:1 omega 7c (14.4%). The sequence similarities of isolate RS1(T) based on gyrB and rpoD genes were 98.9 and 98.0% with Pseudomonas pseudoalcaligenes, and 98.5 and 98.1% with Pseudomonas oleovorans, respectively. The ribotyping pattern showed a 0.60 similarity with P. oleovorans ATCC 8062(T) and 0.63 with P. pseudoalcaligenes ATCC17440(T). The DNA G + C content of isolate RS1(T) was 62.2 mol.%. The DNA-DNA relatedness was 73.0% with P. oleovorans ATCC 8062(T) and 79.1% with P. pseudoalcaligenes ATCC 17440(T). On the basis of morphological, biochemical, and molecular studies, isolate RS1(T) is considered to represent a new subspecies of P. oleovorans. Furthermore, based on the DNA-DNA relatedness (> 70%), chemotaxonomic, and molecular profile, P. pseudoalcaligenes ATCC 17440(T) and P. oleovorans ATCC 8062(T) should be united under the same name; according to the rules of priority, P. oleovorans, the first described species, is the earlier synonym and P. pseudoalcaligenes is the later synonym. As a consequence, the division of the species P. oleovorans into two novel subspecies is proposed: P. oleovorans subsp. oleovorans subsp. nov. (type strain ATCC 8062(T) = DSM 1045(T) = NCIB 6576(T)), P. oleovorans subsp. lubricantis subsp. nov. (type strain RS1(T) = ATCC BAA-1494(T) = DSM 21016(T)).