Murine 8S-lipoxygenase (8S-LOX) is a 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible lipoxygenase. That is, it is not detected in normal mouse skin, however, a significant increase in expression is detected in the skin of TPA promotion-sensitive strains of mice after TPA treatment. In this study, we found TPA-induced 8S-LOX mRNA expression is a result of increased transcription in SSIN primary keratinocytes and further investigated transcriptional regulation of 8S-LOX expression by cloning its promoter. The cloned 8S-LOX promoter ( approximately 2 kb) in which a transcription initiation site was mapped at -27 from the ATG has neither a TATA box nor a CCAAT box. However, the promoter was highly responsive to TPA in TPA promotion-sensitive SSIN but not in TPA promotion-resistant C57BL/6J primary keratinocytes. We then identified a Sp1 binding site located -77 to -68 from the ATG that is a TPA-responsive element (TRE) of the promoter and that Sp1, Sp2, and Sp3 proteins bind to the TRE. We also found that the binding of these proteins to the TRE was significantly increased by TPA treatment and inhibition of the binding by mithramycin A decreased TPA-induced promoter activity as well as 8S-LOX mRNA expression. These data suggest that increased binding of Sp1, Sp2, and Sp3 to the TRE of the 8S-LOX promoter is a mechanism by which TPA induces 8S-LOX expression in keratinocytes.