Hepatic microsomes prepared from rats pretreated with hematoporphyrin derivative (HPD) undergo rapid enhancement of lipid peroxidation in the presence of solar radiation (approximately 400 nm). Quenchers of singlet oxygen, including 2,5-dimethylfuran, histidine, and beta-carotene, and inhibitors of the hydroxyl radical, including benzoate, mannitol, and ethanol, largely protected against the enhancement of lipid peroxidation caused by HPD photosensitization. Catalase, a scavenger of hydrogen peroxide and superoxide dismutase, a scavenger of superoxide anion, had little or no protective effect against HPD-photosensitized enhancement of lipid peroxidation. Our data indicate that in vitro irradiation of hepatic microsomes prepared from HPD-treated rats results in the generation of both singlet oxygen and hydroxyl radical. These reactive moities are associated with a rapid increase in microsomal lipid peroxidation which may explain the unique susceptibility of membranous components of cells to this type of phototoxic injury.