Rod outer segments of vertebrate retinas are highly enriched in docosahexaenoic acid (22:n-3), a fatty acid that is essential for optimal retinal function. The high levels of retinal 22:6n-3 are maintained through conservation (recycling) within the eye and incorporation from the circulation. The liver is thought to be a major source of 22:6n-3 through synthesis from appropriate n-3 precursors and delivery to target tissues via plasma lipoproteins. The contribution of other tissues to the total body pool of 22:6n-3 is not known. We investigated the synthesis of 22:6n-3 from [1-14C]18:3n-3 or [3-14C]22:5n-3 in frog retina and retinal pigment epithelium (RPE). RPE cells rapidly converted each precursor to 22:6n-3, which contained about 23 and 35%, respectively, of the label after 8 h. Significant labeling of 24:6n-3 and 24:5n-3 occurred when [3-14C]22:5n-3 was the substrate. In contrast, the major end products of retinas incubated with [1-14C]18:3n-3 and [3-14C]22:5n-3 were 18:4n-3 and 20:5n-3, respectively, neither of which is found in retinal lipids. Less than 5% of the radioactivity from either precursor was in 22:6n-3 after an 8-h incubation. Our results demonstrate an active in vitro synthesis of 22:6n-3 in frog RPE, but not in the retina. The labeling of 24:5n-3 and 24:6n-3 is consistent with the proposal of Voss et al. [Voss, A., Reinhart, M., Sankarappa, S., & Sprecher, H. (1991) J. Biol. Chem. 266, 19995-20000] that they are intermediates in the conversion of 22:5n-3 to 22:6n-3. Since frog RPE contains measurable amounts of 18:3n-3, 20:5n-3, and 22:5n-3, which are readily converted to 22:6n-3 in these cells, we suggest that the RPE is a source of 22:6n-3 for the retina.