Progression of prostate cancer to lethal forms is marked by emergence of hormone-independent proliferation of the cancer cells. Nutritional and epidemiological studies have indicated that prostate cancer progression is correlated with the consumption of polyunsaturated fatty acids (PUFA). To shed additional light on the cell-level mechanisms of the observed correlation, we compared the sensitivity of hormone-dependent and hormone-independent prostate cancer cells to growth medium supplementation with free PUFAs in a cell proliferation and viability assay. Our data show that the hormone-dependent cells are comparatively insensitive to various PUFAs, at the same time as the growth and viability of hormone-independent cells lines are strongly inhibited by most of the tested PUFAs, whether n-3 or n-6. We speculate that this difference may be at least partially responsible for the observed effects of specific dietary lipids in prostate cancer. The new data strengthen the case for dietary intervention as part of potential new therapeutic strategies seeking to impede prostate cancer progression.