The contribution of lipid peroxidation to the killing of human breast cancer cells by gamma-linolenate (GLA) was examined. Other fatty acids of different cytotoxic potential containing 2, 4, 5, and 6 double bonds were also tested for comparison. It was found that the cytotoxic potential varied with the ability of the fatty acids to stimulate the production of superoxide radicals. Neither hydrogen peroxide nor hydroxy radicals are significantly involved in cell killing. As nonspecific indicators of lipid peroxidation, measurements of the loss of unsaturated fatty acid in the phospholipids together with the generation of hydroperoxide breakdown products were done with the use of the thiobarbituric acid test. The results of these experiments showed that the effectiveness of a given fatty acid in killing cancer cells correlated with the intracellular thiobarbituric acid-reactive material (TBARM) content: GLA and arachidonate with 3 and 4 double bonds generated the most TBARM and were the most cytotoxic fatty acids, whereas docosahexaenoate with 6 double bonds was the least effective either in raising TBARM or in killing the malignant cells. Iron and copper accelerated the rate of cell death, whereas antioxidants such as vitamin E and butylated hydroxyanisole inhibited the effect of GLA dose dependently. Indomethacin, an inhibitor of endoperoxide formation, did not reduce either cell kill or TBARM amounts. In contrast, the addition of vitamin E acetate to the cancer cell cultures challenged with eicosapentaenoate reduced both cell killing and TBARM content. These results suggest that the effectiveness of a given fatty acid in killing cancer cells correlated with the extent of lipid peroxidation of the added fatty acid in the cells.