In higher plants, C6 and C9 aldehydes are formed from C18 fatty acids, such as linoleic or linolenic acid, through formation of 13- and 9-hydroperoxides, followed by their stereospecific cleavage by fatty acid hydroperoxide lyases (HPL). Some marine algae can also form C6 and C9 aldehydes, but their precise biosynthetic pathway has not been elucidated fully. In this study, we show that Laminaria angustata, a brown alga, formed C6 and C9 aldehydes enzymatically. The alga forms C9 aldehydes exclusively from the C20 fatty acid, arachidonic acid, while C6 aldehydes are derived either from C18 or from C20 fatty acid. The intermediates in the biosynthetic pathway were trapped by using a glutathione/glutathione peroxidase system, and subjected to structural analyses. Formation of (S)-12-, and (S)-15-hydroperoxy arachidonic acids [12(S)HPETE and 15(S)HPETE] from arachidonic acid was confirmed by chiral HPLC analyses. These account respectively for C9 aldehyde and C6 aldehyde formation, respectively. The HPL that catalyzes formation of C9 aldehydes from 12(S)HPETE seems highly specific for hydroperoxides of C20 fatty acids.