Eicosanoids, which include prostaglandins, thromboxanes, and leukotrienes, are produced from arachidonic acid by three main pathways in cells, including cyclooxygenases and lipoxygenases, and cytochrome P450 enzymes. Accumulated evidence indicates that a certain peroxide tone is required for the initiation of reaction by lipoxygenases and cyclooxygenases. An endogenous inhibitor of arachidonate oxygenation was suspected in the cytosolic fraction of human epidermoid carcinoma A431 cells. After a series of studies, the existence of this inhibitor was confirmed, while it was purified and characterized. By amino acid sequence analysis, the inhibitor in A431 cells was subsequently identified as a phospholipid hydroperoxide glutathione peroxidase (PHGPx). Depletion of cellular glutathione in cells by diethyl maleate or by dibuthionine-sulfoximine results in an increase in enzyme activities of 12(S)-lipoxygenase and cyclooxygenase, suggesting that glutathione-depleting agents abolish the enzyme activity of PHGPx in cells. Stable transfectants of A431 cells with overexpression and depletion of PHGPx have been constructed, respectively. Reduction of arachidonate metabolism through 12(S)-lipoxygenase and cyclooxygenase 1 and that of the arsenite-induced generation of reactive oxygen species are observed in cells overexpressing PHGPx. On the other hand, enhancement of arachidonate metabolism and the arsenite-induced generation of reactive oxygen species is detected in PHGPx-depleted cells. In conclusion, the endogenous inhibitor of arachidonate metabolism present in A431 cells is a PHGPx, which plays a functional role in the down-regulation of arachidonate oxygenation catalyzed by 12(S)-lipoxygenase and cyclooxygenase 1 through the reduction of the level of intracellular lipid hydroperoxides. The latter acts as the peroxide tone for arachidonate metabolism in A431 cells.