We previously reported that normal human epidermis forms 12-oxo-eicosatetraenoic acid and hepoxilin B3 as major eicosanoids and that hepoxilins and trioxilins are dramatically elevated in psoriatic lesions. We also observed that normal epidermis only synthesized one of the two possible 10-hydroxy- epimers of hepoxilin B3, suggesting its enzymatic origin. This study investigated the enzymatic pathways involved in the formation of hepoxilin B3 in human epidermis. Human epidermal fragments or cell fractions were incubated with [14C]-arachidonic acid or authentic 12(S)-hydroperoxyeicosatetraenoic acid. Products were analyzed by high-performance liquid chromatography, gas chromatography-mass spectrometry or a combination of both techniques. Esculetin and nordihydroguaiaretic acid inhibited formation of hepoxilin B3, 12-oxo-eicosatetraenoic acid, trioxilins, and 12-hydroxyeicosatetraenoic acid in a concentration-dependent manner. 12-Lipoxygenase activity was mainly located in the microsomal fraction (100,000 x g pellet) and 12-hydroxyeicosatetraenoic acid, hepoxilin B3, and 12-oxo-eicosatetraenoic acid were formed. The hepoxilin B3-synthesizing activity was not observed in subcellular fractions incubated with authentic 12(S)-hydroperoxyeicosatetraenoic acid, although it was located at least in the microsomal fraction when incubated with arachidonic acid. Similar results were obtained using preparations of recombinant platelet-type 12-lipoxygenase that yielded 12-oxo-eicosatetraenoic acid and hepoxilin B3 in addition to 12-hydroxyeicosatetraenoic acid, when incubated with arachidonic acid but not when incubated with 12-hydroperoxyeicosatetraenoic acid. Nevertheless, recombinant 12-lipoxygenase produced a lower ratio of 12-oxo-eicosatetraenoic acid and hepoxilin B3-12-hydroxyeicosatetraenoic acid than epidermis. Our results support the concept that 12-lipoxygenase catalyzes the formation of hepoxilin B3 and 12-oxo-eicosatetraenoic acid.