To evaluate the antioxidant effects of 48-carotene and asta-xanthin, rat liver microsomes were exposed to a mixture of chelated iron (Fe³⁺/ADP) and NADPH. The carotenoids (190 pmol/mg protein) were incorporated into some of these microsomal membranes, and phospho-lipid hydroperoxides (PLOOH), thiobarbituric acid reactive substances (TBARS) and endogenous a-tocopherol content were measured over time after the initiation of oxidant stress. In control microsomes, oxidant stress led to accumulation of 1, 865 (+/-371) pmol PLOOH/mg protein during the initial 10-min peroxidation reaction, followed by a more gradual increase during the subsequent 20-min of reaction. PLOOH accumulation during the initial 10-min reaction period was reduced to 588 (+/-169) pmol/mg protein with β-carotene present and 800 (+/-288) pmol/mg protein with astaxanthin present. During the following 20-min of incubation, PLOOH levels declined in the carotenoid-supplemented microsomes but continued to increase at a slower rate in control prepara-tions. TBARS did not show such large accumulation as observed in PLOOH during the initial 10-min incubation in any microsomal sample. The presence of carotenoids in the microsomal membrane partially in-hibited the loss of a-tocopherol, especially during the later phase of oxidant stress. When lipid peroxidation is generated by membrane-bound cyt-P450, the specific measurement of PLOOH clearly demonstrates that the presence of carotenoids provides antioxidant protection.