Cyclooxygenase-2 (COX-2) is important in the progression of epithelial tumors. Evidence indicates that omega-6 PUFAs such as arachidonic acid (AA) promote the growth of tumor cells; however, omega-3 fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] inhibit tumor cell proliferation. We investigated the effects of omega-3 PUFA on the expression and function of COX-2 in 70W, a human melanoma cell line that metastasizes to the brain in nude mice. We show that 1) tumor necrosis factor-alpha upregulates the expression of both COX-2 mRNA and prostaglandin E2 (PGE2) production, and 2) omega-3 and omega-6 PUFA regulate COX-2 mRNA expression and PGE2 production. AA increased COX-2 mRNA expression and prostaglandin production in omega-6-stimulated 70W cells. Conversely, COX-2 mRNA expression decreased in cells incubated with EPA or DHA. AA increased Matrigel invasion 2.4-fold, whereas EPA or DHA did not. Additionally, PGE2 increased in vitro invasion 2.5-fold, whereas exposure to PGE3 significantly decreased invasion. Our results demonstrate that incubation of 70W cells with either AA or PGE2 increased invasiveness, whereas incubation with EPA or DHA downregulated both COX-2 mRNA and protein expression, with a subsequent decrease in Matrigel invasion. Taken together, these results indicate that omega-3 PUFA regulate COX-2-mediated invasion in brain-metastatic melanoma.