Olive oil is an integral ingredient of the "Mediterranean diet" and accumulating evidence suggests that it may have a potential role in lowering risk of several cancers. We recently hypothesized that the anti-cancer actions of olive oil may relate to its monounsaturated fatty acid (MUFA) oleic acid (OA; 18:1n-9) content to specifically regulate oncogenes. In this study, transient transfection experiments with human Her-2/neu promoter-driven luciferase gene established the ability of OA to specifically repress the transcriptional activity of Her-2/neu gene. Gene repression was seen in tumour-derived cell lines with Her-2/neu gene amplification and overexpression, including SK-Br3 (56% reduction), SK-OV3 (75% reduction) and NCI-N87 (55% reduction) breast, ovarian and stomach cancer cell lines, respectively. Also marginal decreases in promoter activity were observed in cancer cells expressing physiological levels of Her-2/neu (20% reduction in MCF-7 breast cancer cells). Remarkably, OA treatment in Her-2/neu-overexpressing cancer cells was found to induce up-regulation of the Ets protein polyomavirus enhancer activator 3 (PEA3), a transcriptional repressor of Her-2/neu promoter. Also, an intact PEA3 DNA-binding-site at endogenous Her-2/neu gene promoter was essential for OA-induced repression of this gene. Moreover, OA treatment failed to decrease Her-2/neu protein levels in MCF-7/Her2-18 transfectants, which stably express full-length human Her-2/neu cDNA controlled by a SV40 viral promoter. OA-induced transcriptional repression of Her-2/neu through the action of PEA3 protein at the promoter level may represent a novel mechanism linking "Mediterranean diet" and cancer.