The supply of docosahexaenoic acid (22:6) to the frog retina, and its subsequent use by retinal cells, was studied by autoradiography and biochemical methods. Different delivery routes of 3H-22:6 were evaluated. Predominant uptake by the neural retina, mainly in ganglion cell axons, outer synaptic layer, and Müller cells, was observed when the radiolabeled fatty acid was given intravitreally or by short-term incubations of eyecups. In short-term eyecup incubations, Müller cells preferentially labeled, suggesting their involvement as a transient storage site. After intravenous or dorsal lymph sac injections of 3H-22:6, most of the retinal label was seen in rod photoreceptor cells. Two different labeling patterns were found in rod outer segments (ROS) as a function of postinjection time: an overall diffuse labeling pattern, as well as a dense-label region at the ROS base. This dense-label region expanded until it reached the apex of the ROS after about 30 d. HPLC analysis of fatty acid methyl esters from retinal lipid extracts showed that 3H-22:6 comprised essentially all of the label until after day 46, indicating lack of metabolic recycling of this molecule. Lipid-extracted retinal residue was devoid of radioactivity, demonstrating that protein did not contain significant covalently bound label. 3H-22:6 acylated to phospholipids in photoreceptor membranes moved apically, as evidenced by the expanding labeled region from the base of the ROS. Oil droplets in both the pigment epithelium and the cone photoreceptors labeled heavily, suggesting that 22:6 may be transiently stored. ROS tips that were phagocytosed by the pigment epithelium contained label similar in density to that of the outer segments, demonstrating that 22:6-phospholipids, at least in part, cycle through the pigment epithelial cells during visual cell renewal. In parallel experiments in frogs injected with 3H-leucine and maintained under the same experimental conditions, well-define, narrow protein bands were observed. Since the leading edge of the 3H-leucine-labeled band (rhodopsin), and that of the dense-label region of 3H-22:6 migrated at the same rate, reaching the rod tips at the same time, we suggest that the 3H-22:6-labeled phospholipids giving this profile are a unique molecular species noncovalently associated with rhodopsin.