Cis-unsaturated fatty acids of both the n-6 and n-3 series have been shown to be cytotoxic to a variety of tumor cells in vitro. Both gamma-linolenic acid (GLA) and eicosapentaenoic acid (EPA) can also augment the cytotoxicity of anticancer drugs. But, the effect of various cis-unsaturated fatty acids on the survival of tumor cells which are resistant to anticancer drugs has not been studied so far. Docosahexaenoic acid (DHA) and EPA of the n-3 series and GLA and dihomo-GLA (DGLA) of the n-6 series were found to be cytotoxic to both vincristine-sensitive (KB-3-1) and resistant (KB-ChR-8-5) human cervical carcinoma (HeLa-variant) cells in vitro. KB-ChR-8-5 was found to be only marginally less sensitive to the cytotoxic actions of all the fatty acids tested except arachidonic acid (AA) compared to KB-3-1. Cyclo-oxygenase inhibitor, indomethacin; lipoxygenase inhibitor, nordihydroguiaretic (NDGA); and calmodulin antagonists, trifluoperazine (TFP) and chlorpromazine (CPZ) were found to be ineffective in blocking the cytotoxic action of GLA, EPA and DHA, the most effective fatty acids, on KB-3-1 cells. This suggests that prostaglandins, leukotrienes and calmodulin do not have any role in the cytotoxic action of GLA, EPA, and DHA. On the other hand, the anti-oxidant, vitamin E, and the superoxide anion quencher, superoxide dismutase (SOD), could completely inhibit the cytotoxic action of GLA, EPA and DHA indicating a role for free radicals and, in particular, the superoxide anion in this process. This was supported further by the observation that GLA, EPA, and DHA can enhance the formation of superoxide anion, hydrogen peroxide, and lipid peroxides in KB-3-1 cells. GLA, EPA, and DHA-induced free radical generation and lipid peroxidation were also inhibited by vitamin E and SOD. These results suggest that GLA, EPA, and DHA are cytotoxic to both vincristine-sensitive and resistant human cervical carcinoma cells and that it is a free radical dependent process.