Primary cultures of smooth muscle cells were established from the medial layer of guinea pig aorta. Confluent cells at passage level 4-6 were challenged with arachidonic acid and treated with a number of antioxidants and inhibitors of specific lipid peroxidation pathways. Lipid peroxidation was measured by the thiobarbituric acid test for malondialdehyde (MDA) and the isolation of hydroperoxy fatty acids (HPETE) by high performance liquid chromatography (HPLC). Prostanoids were measured by radioimmunoassay and the separation of labeled compounds by HPLC. MDA, 6-keto-PGF1 alpha, and PGE2 were formed when cells were challenged with arachidonic acid and these cells synthesized small amounts of one HPETE isomer, 15-HPETE. The HPETE isomers characteristic of the lipoxygenase pathway, 12-HPETE and 5-HPETE, were not detected. Furthermore, the lipoxygenase inhibitors, eicosatetraynoic acid (ETYA) and 6,7-dihydroxycoumarin (Esculetin), did not block MDA formation. These data show that MDA is not generated in the cells by a lipoxygenase pathway. The cyclooxygenase inhibitors, indomethacin and ETYA, did not block MDA formation but these agents blocked the formation of 15-HPETE. These data show both that 15-HPETE is generated by a cooxidation pathway and that 15-HPETE and cooxidation are not involved in MDA formation. Three inhibitors of cytochrome P450 linked lipid peroxidation, 2-amino-3-ethoxycarbonyl-6-benzyl-4, 5,6,7-tetrahydrothieno-[2,3-C]-pyridine (Tinoridine), 3-methyl-1,2-di-3-pyridyl-1-propanone (Metyrapone) and phenobarbital, did not block MDA formation. These data support earlier studies that indicated that MDA is not generated by a P450 pathway. Cells contained a bound precursor that decomposed to MDA when cells were treated with Fe3+. The cells exhibited autofluorescence and concentric lamellae in lipid droplets that are characteristic of ceroid-lipofuscin. These observations are consistent with lipid peroxidation through increased peroxisomal activity leading to the generation of MDA and the accumulation of ceroid-lipofuscin. The natural antioxidants, vitamin E and vitamin E quinone (EQ), and the synthetic antioxidants, butylated hydroxytoluene and nordihydroguaiaretic acid (NDGA), alpha-naphthol (alpha-N) and propyl gallate (PrGa), all blocked MDA formation in confluent smooth muscle cells, showing that these antioxidants did not function solely as specific inhibitors of lipoxygenase, cooxidation or P450 pathways. Cell proliferation was measured in cells challenged with arachidonic acid and treated with antioxidants and other inhibitors. The least cytotoxic and most potent antioxidant, EQ, blocked MDA formation in confluent cells and promoted grow