Although there are many reports of epoxy alcohol synthesis from lipoxygenase products (fatty acid hydroperoxides) in mammalian tissues, there are no well-defined examples of the stereoselective synthesis of individual epoxy alcohol diastereomers. An earlier report on the metabolism of 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE) in rat liver microsomes suggested such a specific reaction [Weiss, R. H., et al. (1987) Arch. Biochem. Biophys. 252, 334-338]. To characterize this reaction further, we set out to determine the precise structures and mechanism of biosynthesis of the epoxy alcohol products. We compared the products formed from 15R- and 15S-HPETE by hematin (a nonenzymatic reaction), by liver microsomes isolated from control and phenobarbital-treated rats, and by purified cytochrome P450 2B1. Eight epoxy alcohol isomers were identified by mass spectrometry and 1H NMR. In the hematin reaction, the major products are four epoxy alcohols with the epoxide in the trans configuration, diastereomers are formed in similar amounts, and the 15-HPETE enantiomers give indistinguishable patterns of products. By contrast, the liver microsomes and P450 2B1 enzyme form predominantly single diastereomers, and the configuration of the epoxide is dependent on the stereochemistry of the substrate. The main product formed from 15S-HPETE is 11S-hydroxy-14S,15S-trans-epoxyeicosa-5Z,8Z,12E- trienoic acid, and the amounts increase upon phenobarbital induction. The main products from 15R-HPETE are 11-hydroxy-14S,15R-epoxyeicosa-5Z,8Z,12E-t rienoic acid from microsomes from control rats and 13-hydroxy-14S,15R-cis-epoxyeicosa-5,8,11-trienoic acid in microsomes from phenobarbital-induced rats. The P450 2B1 enzyme gave products similar to those from the phenobarbital-induced microsomes. Analysis of an incubation using the 18O-labeled 15S-HPETE substrate demonstrated 97.6% retention of both hydroperoxy oxygens in the major product with progressively lower 18O retentions in the minor products (74-32%), possibly reflecting degrees of enzymatic control of these reactions. These results establish a precedent for the stereoselective synthesis of epoxy alcohols by mammalian cytochrome P450s.