The allene oxide synthase (hydroperoxide dehydrase) of flaxseed is a cytochrome P450 that exhibits an exceptionally high catalytic turnover (> or = 1000/s) for hydroperoxy substrates. In a previous study, using a crude extract of flaxseed, we detected a secondary activity that could offer an insight into the mechanism of the enzymatic transformation of hydroperoxides. We observed that the substrate 8R-hydroxy-15S-hydroperoxyeicosa-5,9,11,13,17-pentaenoic acid is converted not only to allene oxide, but also to epoxyalcohol derivatives (Brash, A. R., Baertschi, S. W., and Harris, T. M. (1990) J. Biol. Chem. 265, 6705-6712). The transformation of hydroperoxides to epoxyalcohols has been investigated extensively in other systems, and heterolytic or homolytic cleavage of the hydroperoxide is associated with characteristic rearrangements and stereochemistry of the epoxyalcohol products. Using the purified enzyme, we established that the epoxyalcohols are products of the allene oxide synthase. Their structures were determined by UV, gas chromatography-mass spectrometry, and NMR. The major epoxyalcohol is 8R,13R-dihydroxy-14R,15S-epoxyeicosa-5Z,9E ,11Z,17Z-tetraenoic acid, a trans-epoxide with an alpha-hydroxyl in the relative threo configuration. Two minor products are the corresponding 11E isomer and a cis-epoxide identified as 8R,13-dihydroxy-14S,15S-epoxyeicosa-5Z,9E,11E,++ +17Z-tetraenoic acid. Gas chromatography-mass spectrometry analysis of a reaction with [18O2]hydroperoxide substrate indicated complete retention of the hydroperoxy oxygens in the epoxyalcohol products. Mechanistic precedents support a homolytic hydroperoxide cleavage as the initial step in the synthesis of these epoxyalcohols. We suggest that the same process initiates allene oxide synthesis, a conclusion that is also most compatible with the known chemistry of cytochromes P450.