Cytochrome P450 (P450) 2U1 has been shown to be expressed, at the mRNA level, in human thymus, brain, and several other tissues. Recombinant P450 2U1 was purified and used as a reagent in a metabolomic search for substrates in bovine brain. In addition to fatty acid oxidation reactions, an oxidation of endogenous N-arachidonoylserotonin was characterized. Subsequent NMR and mass spectrometry and chemical synthesis showed that the main product was the result of C-2 oxidation of the indole ring, in contrast to other human P450s that generated different products. N-Arachidonoylserotonin, first synthesized chemically and described as an inhibitor of fatty acid amide hydrolase, had previously been found in porcine and mouse intestine; we demonstrated its presence in bovine and human brain samples. The product (2-oxo) was 4-fold less active than N-arachidonoylserotonin in inhibiting fatty acid amide hydrolase. The rate of oxidation of N-arachidonoylserotonin was similar to that of arachidonic acid, one of the previously identified fatty acid substrates of P450 2U1. The demonstration of the oxidation of N-arachidonoylserotonin by P450 2U1 suggests a possible role in human brain and possibly other sites.