Selective removal of intracellular glutathione (GSH) and inhibition of the GSH-dependent peroxidase (GSH-Px) by 1-chloro-2,4-dinitrobenzene (CDNB) was used to evaluate the role of GSH and GSH-Px in arachidonic acid (AA) metabolism in human platelets. Although total conversion of AA through the lipoxygenase pathway is lowered by GSH depletion, significant 12-HETE formation was observed suggesting that GSH and GSH-Px are not required for the generation of 12-HETE in human platelets. Prolonged treatment of platelets with CDNB (2 h) completely destroyed GSH-Px activity creating a model in which the effects of GSH alone could be determined. Platelet homogenates replenished with GSH, but lacking GSH-Px activity converted significantly higher amounts of AA to 12-HPETE and 12-HETE than control. Platelet cytosolic metabolism of 15-HPETE to 15-HETE decreased after CDNB, while the membrane metabolism remained similar to control due to high GSH-independent peroxidase activity associated with the membranes. These results indicate that GSH and GSH-Px function to enhance lipoxygenase activity, rather than catalyse the reduction of 12-HPETE to 12-HETE.