To investigate the involvement of prostaglandin H synthase (PHS) and lipoxygenase (LPO) in the activation of xenobiotics in platelets, platelet sonicates were preincubated with alpha-naphthol. Protein covalent binding of alpha-naphthol was measured following addition of arachidonic acid. Protein covalent binding was increased in a dose-dependent manner until it plateaued at 500 microM arachidonic acid. Pretreatment by two inhibitors of PHS, aspirin and indomethacin, resulted in a dose-dependent inhibition of alpha-naphthol-induced covalent binding, confirming PHS involvement. In addition, pretreatment by a LPO inhibitor, nordihydroguaiaretic acid (NDGA), also prevented covalent binding substantially, showing that LPO may be an alternative pathway for xenobiotic activation in platelets. Furthermore, combined treatment of aspirin and NDGA almost abolished the increase of alpha-naphthol-induced covalent binding, suggesting that PHS and LPO are both major pathways for xenobiotic activation in platelets.