It has been suggested that exogenous unsaturated fatty acids (UFAs) may increase the cytotoxic activity of cancer chemotherapeutic agents. We examined how y-linolenic acid (GLA; 18: 3n-6), the most promising UFA in the treatment of human tumors, affects the effectiveness of the lipophilic drug vinorelbine (VNR) on human breast carcinoma cell lines. Cells were exposed simultaneously to VNR and GLA or sequentially to GLA followed by VNR. Cell viability was determined by MTT assay. The increase in VNR-induced cell growth inhibition was measured by dividing the IC50 and IC70 values (50 and 70% inhibitory concentrations, respectively) that were obtained when the cells were exposed to VNR alone with those with VNR plus GLA. We found that GLA enhanced in a dose-dependent manner the cell growth inhibitory activity of VNR on MCF-7 cells (up to 9-fold). As GLA by itself showed anti-proliferative effects, possible GLA-VNR interactions at the cellular level were assessed employing the isobologram analysis and the combination index (CI) method of Chou-Talalay. Both methods showed an overall synergism between GLA and VNR in MCF-7 cells. At a high level of cell kill, the synergism was greater when a 24 h GLA pre-exposure or co-exposures were tested. Synergy was likewise observed with the GLA-VNR combination in MDA-MB-231, T47D, and SK-Br3 breast cancer cells. In all cell lines, the synergism was independent of the treatment schedule and the exposure time. Under conditions inhibiting lipid peroxidation using Vitamin E (dl-alpha-tocopherol), the enhancing effect of GLA (an easily oxidizable UFA) on VNR activity was partially abolished. However, when Vitamin E was used in combination, a similar synergistic increase in growth inhibition was obtained. These latter observations strongly implies that the synergistic effects of GLA with VNR are not mediated through a mechanism involving a generation of lipoperoxides. For comparison, the effects of other UFAs were examined on VNR chemosensitivity: GLA was the most potent at enhancing VNR activity, followed by docosahexaenoic acid (22: 6n-3), eicosapentaenoic acid (20: 5n-3) and alpha-linolenic acid (18: 3n-3), whereas linoleic acid (18: 2n-6) and arachidonic acid (20: 4n-6) did not increase VNR chemosensitivity. Very high concentrations of oleic acid (OA; 18:1 n-9), an UFA inversely correlated with breast cancer risk, also enhanced VNR effectiveness. Thus, various types of UFAs were not equivalent with respect to their actions on VNR effectiveness. In conclusion, our results give experimental support to the hypothesis that some UFAs can be used as modulators of tumor cell chemosensitivity and provide the rationale for in vivo preclinical investigation.