Dietary intake of essential fatty acids (EFA) may play a role in prostate cancer cell proliferation. Epidemiological studies have demonstrated that men whose dietary intake is high in omega-3 fatty acid (FA) composition have a lower incidence of clinical prostate cancer, suggesting that external factors such as diet may play an important role in development and growth of prostate cancer. Furthermore, in prostate cancer cell lines, omega-6 and omega-3 FAs have demonstrated promotional and inhibitory effects respectively. To investigate the effects of dietary fats on nontumorigenic prostate cell growth we conducted in vitro studies with human metastatic PC-3, LNCaP and TSU prostate cell lines, the rat metastatic Mat-Ly-Lu cell line and rat non-metastatic epithelial cell lines EPYP1, EPYP2 and EPYP3. Cell lines were treated with linoleic acid (LA), an omega-6 FA (n-6), as well as linolenic (LLA) and eicosapentaenoic (EPA) acids, which are both omega-3 FAs (n-3). All cell lines were treated with 10% and 0.5% serum supplemented media plus fatty acid for comparison. Our results demonstrate that linoleic acid(n-6) has promotional effects at doses of 1-100ng/ml in all cell lines with the exception of EPYPl. Experiments with linolenic acid (n-3) demonstrated consistent growth promotion in all cell lines examined with the exception of the EPYP2 cell line in which there was no significant effect. EPA had no effect in culture media supplemented with 10% serum, while in media containing 0.5% serum this FA demonstrated significant promotion in all human lines. Previous studies have indicated that EPA should inhibit human prostate cancer growth in vitro, however our results demonstrated promotion at low concentrations (lng/ml). At higher concentrations, EPA did inhibit prostate cell growth. These data indicate low levels of dietary fat, regardless of composition, may play a role in prostate cancer proliferation and could be an avenue for therapeutic intervention.