Human platelets possess active lipoxygenase and cyclooxygenase which convert arachidonic acid to (12S)-12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) plus (12S)-12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and thromboxane B2 plus 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), respectively. When platelet homogenates were incubated with arachidonate, there was a rapid consumption of platelet tocopherol. Time course analysis revealed that within 0.5 min, over half of arachidonate and tocopherol were metabolized. Mass formation of 12-HPETE and 12-HETE or thromboxane B2 and HHT exceeded that of the mass of tocopherol oxidized. Preincubation with the lipoxygenase inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) completely abolished this arachidonate-induced tocopherol oxidation whereas cyclooxygenase inhibitors (indomethacin and aspirin) further potentiated tocopherol oxidation, indicating that this oxidation is closely linked with platelet 12-lipoxygenase activity. Incubation with lipoxygenase metabolites of arachidonic acid showed that only 12-HPETE caused a rapid tocopherol oxidation which was followed by a gradual tocopherol regeneration. By using nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor which is also a strong reductant, over 60% of the arachidonate-induced oxidized tocopherol was regenerated. Tocopherol regeneration declined with increasing oxidation time induced by arachidonate, and after 30-60 min virtually no regeneration could be observed, suggesting that the precursor molecule was unstable. We postulate that the precursor molecule is the tocopheroxyl radical. In the presence of ETYA, a lipoxygenase inhibitor without antioxidant properties, either ascorbate or GSH provided significant tocopherol regeneration. Kinetic studies showed that tocopherol regeneration after the addition of ascorbate was essentially completed by 1 min. By contrast, GSH addition caused a steady increase in tocopherol which peaked after 10 min of its addition. To determine whether this rapid regeneration is chemical or enzymic, regeneration was studied in the presence of chloroform and methanol. Comparison of various reductants in this denaturing condition for enzymes showed that ascorbate and NDGA afforded significant regeneration whereas GSH was ineffective, indicating that there are distinct enzymic and non-enzymic mechanisms for tocopherol regeneration. This study provides direct evidence from mass analysis that tocopherol can be regenerated in human cell homogenates. This finding implies that maintenance of membrane tocopherol status may be an essential function of ascorbate and GSH which operate in concert to ensure maximum membrane protection against oxidative damage.