We investigated the catalytic and kinetic properties of allene oxide synthase (AOS; E.C. 4.2.1.92) from flaxseed (Linum usitatissimum L.). Both Michaelis constant and maximal initial velocity for the conversion of 9(S)- and 13(S)-hydroperoxides of linoleic and linolenic acid were determined by a photometric assay. 13(S)-Hydroperoxy-9(Z), 11(E)-octadecadienoic acid [13(S)-HPOD] as the most effective substrate was converted at 116.9 +/- 5.8 nkat/mg protein by the flax enzyme extract. The enzyme was also incubated with a series of variable conjugated hydroperoxy dienyladipates. Substrates with a shape similar to the natural hydroperoxides showed the best reactivity. Monoenoic substrates as oleic acid hydroperoxides were not converted by the enzyme. In contrast, 12-hydroperoxy-9(Z), 13(E)-octadecadienoic acid was a strong competitive inhibitor for AOS catalyzed degradation of 13(S)-HPOD. The inhibitor constant was determined to be 0.09 microM. Based on these results, we concluded that allene oxide synthase requires conjugated diene hydroperoxides for successful catalysis. Studying the enantiomeric preference of the enzyme, we found that AOS was also able to metabolize (R)-configurated fatty acid hydroperoxides. Conversion of these substrates into labile allene oxides was confirmed by steric analysis of the stable alpha-ketol hydrolysis products.