12-lipoxygenase-catalyzed arachidonic acid metabolism in normal and neoplastic mouse epidermis was assessed by cDNA cloning of the epidermal 12-lipoxygenases and by studying their expression patterns, enzyme activities, and product levels. Papillomas and squamous cell carcinomas induced by the initiation/promotion protocol contained 50- to 60-fold more 12-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) than normal epidermis. The ratio of S to R enantiomers was 9:1. This indicates that most of this eicosanoid was of enzymatic origin. Accordingly, cell-free preparations of the tumors exhibited about fivefold elevated 12-lipoxygenase activities. A papilloma-derived cDNA library was screened with human platelet-type 12-lipoxygenase cDNA probes. Two cDNA clones encoding the platelet-type and the leukocyte-type isoforms of murine 12-lipoxygenase were isolated, demonstrating the coexpression of the isoenzymes in the same tissue and species. When expressed in COS-7 cells, the recombinant enzymes showed the characteristic substrate selectivity and product profile, with the leukocyte-type enzyme metabolizing linoleic and arachidonic acid to 13-hydroxy-9,11-octadecadienoic acid and to 12- and 15-HETE, respectively, and the platelet-type enzyme oxygenating exclusively arachidonic acid to 12-HETE. In epidermis in vivo and in keratinocytes in culture, only the platelet-type 12-lipoxygenase (mRNA and protein) was detectable. In mouse epidermis both isoenzymes were induced transiently by phorbol esters. Most tumors showed constitutive overexpression of platelet-type mRNA, whereas leukocyte-type specific transcripts were detectable only in a few tumors. These data suggest that the platelet-type enzyme is the 12-lipoxygenase isoform of keratinocytes that is responsible for the generation of most of the 12-HETE found in neoplastic epidermis.