The microsomal fraction of mouse liver homogenate showed NAD(P)-dependent dehydrogenase activity involved in the conversion of 15-hydroxyeicosatetraenoic acid to 15-ketoeicosatetraenoic acid, which was determined quantitatively by HPLC assay. This enzyme, tightly bound to membranes and relatively stable, possessed apparent values of Km of 8.3 microM and Vmax of 2.8 nmoles/mg.min in the oxidation of 15-HETE, and gave an optimum pH of 9.8. Additionally, the enzyme, not susceptible to the inhibition by indomethacin and showing a similar cosubstrate specificity between NAD and NADP, utilized other hydroxylated eicosanoids as substrates, based on HPLC analyses.