The levels of 8S-lipoxygenase (8S-LOX) expression and of its arachidonic acid metabolite, 8-hydroxyeicosatetraenoic acid (8-HETE), are highly elevated in the early stages of mouse skin carcinogenesis. On the other hand, several reports showing that 8-HETE is also closely associated with keratinocyte differentiation raise a question concerning the role of 8S-LOX/8-HETE in skin carcinogenesis. To address that question, here we conducted a series of gain-of-function studies. Skin targeted loricrin 8S-LOX/C57BL/6J transgenic mice showed a more differentiated epidermal phenotype as well as a 64% reduced papilloma development in a two-stage skin carcinogenesis protocol. Forced expression of 8S-LOX in MT1/2 cells, a murine papilloma cell line, also caused a more differentiated appearance as well as keratin 1 expression. Overexpression of 8S-LOX in CH72 cells, a murine carcinoma cell line, inhibited cell proliferation by 30% in vitro and by 86% in in vivo xenografts. Exogenous addition of 5 muM 8-HETE to CH72 cells caused cell cycle arrest at the G1 phase. Finally, immunohistochemical analyses showed 8S-LOX protein expression was strictly confined to the differentiated compartment of mouse skin and throughout tumorigenesis. Collectively, these data suggest that 8S-LOX plays a role as a prodifferentiating, antitumorigenic, and tumor suppressing gene in mouse skin carcinogenesis.