Recent studies on jasmonic acid (JA) biosynthetic mutants have shown that jasmonates play essential roles in pollen maturation and dehiscence and wound-induced defence against biotic attacks. To better understand the biosynthetic mechanisms of this essential plant hormone, we isolated an Arabidopsis knock-out mutant defective in the JA biosynthetic gene CYP74A (allene oxide synthase, AOS) using reverse genetics screening methods. This enzyme catalyses dehydration of the hydroperoxide to an unstable allene oxide in the JA biosynthetic pathway. Endogenous JA levels, which increase 100-fold 1 h after wounding in wild-type plants, do not increase after wounding in the aos mutant. In addition, the mutant showed severe male sterility due to defects in anther and pollen development. The male-sterile phenotype was completely rescued by exogenous application of methyl jasomonate and by complementation with constitutive expression of the AOS gene. RT-PCR analysis showed that the induction of transcripts for vegetative storage protein and lipoxygenase genes, previously shown to be inducible by wound and jasmonate application in the wild-type, was absent in the aos mutant. In transgenic plants constitutively expressing AOS, wound-induced JA levels were 50-100% higher compared to wild-type plants. Taken together with JA deficiency in the aos mutant, our results show that AOS is critical for the biosynthesis of all biologically active jasmonates. Our results also suggest that AOS expression is limiting JA levels in wounded plants, but that the AOS hydroperoxide substrate levels, controlled by upstream enzymes (lipoxygenase and phospholipase), determine JA levels in unwounded plants.