Lipoxygenase was purified homogeneously from cups of Pleurotus ostreatus by Sephacryl S-400 HR gel filtration, Dyematrex Green A affinity, and DEAE-Toyopearl 650M ion-exchange chromatographies. The molecular weight of the enzyme was estimated to be 67 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 66 000 by gel filtration; the isoelectric point was pH 5.1. The optimum pH and temperature of the enzymatic activity were 8.0 and 25 degreesC, respectively. The enzyme contained non-heme iron, and a thiol group seemed to be involved in its activity. The K-m, V-max and k(cat) values of the enzyme for linoleic acid were 0.13 mM, 23.4 mumol(.)min(-1.)mg(-1), and 25.7 s(-1), respectively. The enzyme showed high specificity toward linoleic acid. When linoleic acid was incubated with the enzyme, 13-hydroperoxy-9Z, 11E-octadecadienoic acid was found to be the main oxidative product.