Vertebrate retinas, especially photoreceptor cellular membranes, contain high levels of docosahexaenoic acid (DHA) and relatively low levels of arachidonic acid (AA). The present study was designed to test the hypothesis that DHA enrichment in the retina is the result of preferential uptake and incorporation relative to other fatty acids. Frog retinas were incubatedin vitrowith [3H]DHA or [3H]AA for up to 6 h, and the amounts of label in retinal lipids were quantitated. The incorporation of DHA into total lipids, triglycerides, phosphatidylcholine, and phosphatidylethanolamine was similar to that of AA when each was presented as the only substrate, and was linear with fatty acid concentration and incubation time. The addition of excess, unlabeled AA reduced the uptake and incorporation of DHA into retinal lipids. A slightly greater inhibition was noted for the uptake and incorporation of AA in the presence of unlabeled DHA. There was about 2-3 fold greater incorporation of DHA into phosphatidic acid, diglycerides, and phosphatidylinositol compared with AA, whereas the reverse was found for phosphatidyiserine. The different levels of DHA and AA in retinal phospho-lipids cannot be explained by different rates of uptake and incorporation of these fatty acids into lipids, although some slight enrichment of DHA may be possible by this mechanism. The linear incorporation with fatty acid concentration suggests that the difference could be accomplished by controlling the amount and type of fatty acids delivered to the retina by the adjacent pigment epithelium.