Di-(2-ethylhexyl) phthalate (DEHP), a peroxisome proliferator-activated receptor alpha (PPARalpha) ligand, alters the lipid composition of rat testis, yet the mechanism is unclear. In this study, we investigated the effect of DEHP on the synthesis and metabolism of arachidonic acid (AA), a precursor of eicosanoids, in the testis of prepubertal rats. DEHP (100 and 1,000 mg/kg, 5 days) administration caused a significant reduction in activity of cytosolic phospholipase A2 (cPLA2), the rate-limiting enzyme in the AA and eicosanoid synthesis pathways. DEHP increased the expression of 12-lipoxygenase (12-LOX) in rat testis, whereas cyclooxygenase-2 (COX-2) expression was not altered. Cytochrome P450 4A1 (CYP4A1), a product of a PPARalpha-regulated gene, was markedly increased in the testis by DEHP administration. Taken together, DEHP suppresses cPLA2 activity and induces the AA metabolizing enzymes such as 12-LOX and CYP4A1, resulting in the reduction of AA level. These data suggest that altered AA metabolic cascades may be related to the decrease of testosterone concentration in DEHP-induced testicular atrophy.