: We investigated the mechanism by which rat retina conserves docosahexaenoic acid during essential fatty acid deficiency. Weanling female albino rats were fed diets containing either 10% by weight hydrogenated coconut oil, safflower oil, or linseed oil for 15 weeks. Plasma and rod outer segment (ROS) membranes were prepared for fatty acid and phospholipid molecular species analysis. In addition, retinas were removed for morphometric analysis. We found the following: (1) Plasma phospholipids and cholesterol esters from coconut oil, safflower oil, and linseed oil diet groups were enriched in 20:3(n‐9), 20:4(n‐6), and 20:5(n‐3), respectively. The levels of these 20‐carbon fatty acids in the ROS, however, were only slightly affected by diet. (2) The fatty acids and molecular species of ROS phospholipids from the safflower oil and coconut oil groups showed a selective replacement of 22:6(n‐3) with 22:5(n‐6), as evidenced by a reduction of the 22:6(n‐3)‐22:6(n‐3) molecular species and an increase in the 22:5(n‐6)‐22:6(n‐3) species. (3) The renewal rate of ROS integral proteins, determined by autoradiography, was 10% per day for each diet group. (4) Morphometric analysis of retinas showed no differences in the outer nuclear layer area or in ROS length between the three groups. We conclude that the conservation of 22:6(n‐3) in ROS is not accomplished through reductions in the rate of membrane turnover, the total amount of ROS membranes, or in the number of rod cells. The retina may conserve 22:6(n‐3) through recycling within the retina or between the retina and the pigment epithelium, or through the selective uptake of 22‐carbon poly‐unsaturated fatty acids from the circulation.