Our previous studies have shown that alpha-eleostearic acid (alpha-ESA; cis-9, trans-11, trans-13 (c9,t11,t13)-conjugated linolenic acid (CLnA)) is converted into c9411-conjugated linoleic acid (CLA) in rats. Furthermore, we have demonstrated that the conversion of alpha-ESA into CLA is a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzymatic reaction, which occurs mostly in the rat liver. However, the precise metabolic pathway and enzyme involved have not been identified yet. Therefore, in this study we aimed to determine the role of cytochrome P450 (CYP) in the conversion of alpha-ESA into c9-t11-CIA using an in vitro reconstitution system containing mouse hepatic microsomes, NADPH, and alpha-ESA. The CYP4 inhibitors, 17-ODYA and HET0016, performed the highest level of inhibition of CLA formation. Furthermore, the redox partner cytochrome P450 reductase (CPR) inhibitor, 2-chloroethyl ethyl sulfide (CEES), also demonstrated a high level of inhibition. Thus, these results indicate that the NADPHdependent CPR/CYP4 system is responsible for CLA formation. In a correlation analysis between the specific activity of CLA formation and Cyp4 family gene expression in tissues, Cypla14 and Cyp-f13 demonstrated the best correlations. However, the CYP4F substrate prostaglandin A(1) (PGA(1)) exhibited the strongest inhibitory effect on CLA formation, while the CYP4A and CVP4B1 substrate lauric acid had no inhibitory effect. Therefore, we conclude that the CYP4F13 enzyme is the major enzyme involved in CLA formation. This pathway is a novel pathway for endogenous CLA synthesis, and this study provides insight into the potential application of CLnA in functional foods.