Fatty acid hydroperoxides formed by lipoxygenase can be cleaved by hydroperoxide lyase resulting in the formation of short-chain aldehydes and omega-oxo acids. Plant hydroperoxide lyases use 13- or 9-hydroperoxy linoleic and linolenic acid as substrates. Alfalfa (Medicago sativa L.) has been reported to contain a hydroperoxide lyase specific for 13-hydroperoxy linoleic and linolenic acid only. However, in addition to 13-hydroperoxide lyase activity we found substantial 9-hydroperoxide lyase activity in alfalfa seedlings as well. The specific activity for 9-hydroperoxy fatty acids was about 50% of the activity for the 13-isomers. Furthermore, alfalfa seedlings contain a 3Z:2E-enal isomerase that converts the 3Z-enal products to their 2E-enal isoforms.