High levels of n-6 docosapentaenoic acid (22:5n-6) have been reported in the retina of guinea pigs fed commercially-prepared grain-based rations (commercial diet). In rats and monkeys, high levels of 22:5n-6 are an indicator of n-3 polyunsaturated fatty acid (PUFA) deficiency. We have examined the fatty acid composition of the retina and brain in guinea pigs fed a commercial diet or one of three semi-purified diets containing three different levels of n-3 PUFA. The diets comprised a diet deficient in n-3 PUFA (semi-purified diet containing safflower oil), two diets containing α-linolenic acid (standard commercial laboratory diet and semi-purified diet containing canola oil), and a diet containing α-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid (DHA) (semi-purified diet containing canola oil, safflower oil, and fish oil). Two groups of guinea pigs were given the diets from day 1 to 4 wk or day 1 to 8 wk, when they were sacrificed and the retinal tissues were extracted and analyzed for PUFA content by gas-liquid chromatography. Fatty acid analyses of the retinal phospholipids of the four-week-old animals revealed that the group fed DHA (from the fish oil) had the highest level of DHA (32%), compared with values of 19 and 13% for the groups fed canola oil diet and commercial diet, respectively, and 2% for the group fed the diet deficient in n-3 PUFA. The levels of 22:5n-6 in the retinal lipids were inversely related to the DHA values, being 0.6, 6.6, 11.4, and 20.6 for the fish oil, canola oil, commercial diet, and safflower oil diet groups, respectively. The long-chain PUFA profiles in the brain phospholipids of the four-week-old group were similar to those from the retina. The retinal PUFA values for the eight-week-old animals were similar to the four-week-old group. The safflower oil diet induced a greater deficit of DHA in retinal lipids than has been reported in rats and monkeys fed similar diets. The guinea pigs fed the commercial diet had retinal and brain PUFA patterns similar to that produced by n-3 PUFA-deficient diets in rats and monkeys. Guinea pigs fed the canola oil diet had significantly greater retinal DHA levels than those fed the commercial diet, but lower than those fed fish oil. The data suggest that the guinea pig has a reduced capacity for DHA synthesis from α-linolenic acid as compared with other mammals. Supplementation of guinea pig diets with fish oil produced high retinal and brain DHA levels and prevented the accumulation of 22:5n-6.