The oxidative breakdown of linoleic acid leading to the formation of the mushroom flavor alcohol 1-octen-3-ol by mycelial homogenate of the edible mushroom Pleurotus pulmonarius grown in submerged culture was studied The compounds 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid (13-HPOD) and 10-oxo-trans-8-decenoic acid (10-oxo-acid) were found to be the major nonvolatile metabolites associated with the enzymatic cleavage of linoleic acid to 1-octen-3-ol. Whereas 1-octen-3-ol and 10-oxo-acid were produced at all linoleic acid concentrations studied (up to 8 mM), 13-HPOD was absent at linoleic acid concentrations below 1 mM but became the major nonvolatile product of the enzymatic oxidation at concentrations above 1 mM. Despite its accumulation, 13-HPOD was found not to be the precursor of 1-octen-3-ol when it was supplied as the substrate instead of linoleic acid. Periodic addition of linoleic acid during the course of the reaction maintained 1-octen-3-ol formation at a constant rate while 13-HPOD accumulated, suggesting that 1-octen-3-ol formation may be limited by either competitive product inhibition or substrate availability. These results suggest the involvement of two different lipoxygenases in 1-octen-3-ol and 13-HPOD formation. Mild heat treatment of the pellets before homogenization and incubation completely inhibited 1-octen-3-ol formation and resulted in the accumulation of an additional HPOD, possibly a 1-octen-3-ol precursor. It would appear, therefore, that 13-HPOD accumulation takes place in parallel with 1-octen-3-ol and 10-oxo-acid biosynthesis, suggesting that these are two distinct biosynthetic pathways catalyzed by two different lipoxygenases.