The purpose of this study was to determine whether dietary n-3 and n-6 PUFA may affect retinal PUFA composition and PGE1 and PGE2 production. Male Wistar rats were fed for 3 months with diets containing: (1) 10% eicosapentaenoic acid (EPA) and 7% docosahexaenoic acid (DHA), or (2) 10% γ-linolenic acid (GLA), or (3) 10% EPA, 7% DHA and 10% GLA, or (4) a balanced diet deprived of EPA, DHA, and GLA. The fatty acid composition of retinal phospholipids was determined by gas chromatography. Prostaglandin production was measured by enzyme immunoassay. When compared to rats fed the control diet, the retinal levels of DHA were increased in rats fed both diets enriched with n-3 PUFA (EPA + DHA and EPA + DHA + GLA diets) and decreased in those supplemented with n-6 PUFA only (GLA diet). The diet enriched with both n-6 and n-3 PUFA resulted in the greatest increase in retinal DHA. The levels of PGE1 and PGE2 were significantly increased in retinal homogenates of rats fed with the GLA-rich diet when compared with those of animals fed the control diet. These higher PGE1 and PGE2 levels were not observed in animals fed with EPA + DHA + GLA. In summary, GLA added to EPA + DHA resulted in the highest retinal DHA content but without increasing retinal PGE2 as seen in animals supplemented with GLA only.