Human cytochrome P450 (P450) 2W1 is still considered an "orphan" because its physiological function is not characterized. To identify its substrate specifi city, the purifi ed recombinant enzyme was incubated with colorectal cancer extracts for untargeted substrate searches using an LC/ MS-based metabolomic and isotopic labeling approach. In addition to previously reported fatty acids, oleyl (18:1) lysophosphatidylcholine (LPC, lysolecithin) was identifi ed as a substrate for P450 2W1. Other human P450 enzymes tested showed little activity with 18:1 LPC. In addition to the LPCs, P450 2W1 acted on a series of other lysophospholipids, including lysophosphatidylinositol, lysophosphatidylserine, lysophosphatidylglycerol, lysophosphatidylethanolamine, and lysophosphatidic acid but not diacylphospholipids. P450 2W1 utilized sn -1 18:1 LPC as a substrate much more efficiently than the sn -2 isomer; we conclude that the sn -1 isomers of lysophospholipids are preferred substrates. Chiral analysis was performed on the 18:1 epoxidation products and showed enantio-selectivity for formation of (9 R ,10 S ) over (9 S ,10 R ). Although the genomic sequences of human and numerous other organisms have been established, the functions of less than one-half of the proteins have been annotated, even in Escherichia coli . Thus, an important and challenging