Omega-6 (n-6) polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA), are associated with promoting the progression of prostate cancer, whereas n-3 PUFA, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) tend to exert the opposite effect. Cell migration and invasion can be potentially altered after incorporation of these PUFA into the cellular membrane. The aim of this project was to investigate the effects of AA, EPA and DHA on the cellular membrane composition and explore the effects of these treatments and membrane modifications on prostate cancer metastatic potential. Four different prostate cell lines (PNT2, LNCaP, VCaP and PC-3) with different responses to androgens were used in this project and cells were treated with AA, DHA or EPA (5-50 ÃÂ¼M). Growth rates, glycolysis and respiration function of these cells were characterized. Cell toxicity was assessed using the MTT and SRB assays, cellular fatty acids and lipids were assessed by gas chromatography, liquid chromatography coupled to tandem mass spectrometry and ultra-high performance supercritical fluid chromatography to mass spectrometry. Cell migratory and invasion potential were assessed using a modified scratch assay and Matrigel-coated invasion chambers, respectively. The expression of CD44 and CD36 proteins was assessed by flow cytometry. The non-malignant PNT2 cell line showed the fastest growth rate, followed by the androgen-sensitive LNCaP cell line, then androgen-insensitive PC-3 cells and lastly, the androgen-sensitive VCaP cell line. AA, EPA and DHA had very similar effects on cell proliferation. The androgen-sensitive LNCaP and VCaP cells exhibited the same growth pattern towards these fatty acids: their growth was slightly inhibited. The androgen-insensitive PNT2 and PC-3 cells showed similar growth patterns towards these fatty acids, which inhibited cell growth to a higher extent. The inhibitory effect of these fatty acids on PC-3 cell proliferation was associated with suppression of cellular metabolic function. The aggressive PC-3 cells and the non-malignant PNT2 cells exhibited higher fatty acid accumulation and higher lipid mediator production compared with the androgen-sensitive LNCaP and VCaP cells, following treatment with PUFA. High levels of cholesteryl ester (CE) and palmitic acid (PALM) were observed in PC-3 cells compared to the other cell lines. This profile maybe linked to the aggressiveness of this cell line. AA, EPA and DHA were esterified mainly to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species with higher preference for the PE. The incorporation of PUFA was higher in PNT2 and PC-3 cells. Only DHA treatment reduced PE levels in all cell lines. AA enhanced the invasive capacity of PC-3 cells and this was associated with long-term up-regulation of CD44 protein. DHA inhibited PC-3 cell migratory potential and this was associated with short-term suppression of pro-metastatic CD44 protein expression. EPA did not have any effect on PC-3 cell migration and CD44 expression, but it induced PC-3 cell invasion. Pre-treatment of PC-3 cells with PUFA inhibited invasion when AA and EPA were used as chemoattractants, and this was associated with reduced levels of cellular oleic acid and monounsaturated fatty acids. Overall, AA may enhance prostate cancer progression, potentially by promoting the invasive capacity of prostate cancer cells and up-regulating the pro-metastatic CD44 protein expression. DHA may prevent prostate cancer progression by inhibiting cell migratory potential, suppressing CD44 protein expression and reducing membrane PE levels. These changes may be leading to disruption of lipid rafts resulting in changes in cell migration and invasion.