OBJECTIVE To determine if soluble binding proteins (BP) for fatty acids (FA), particularly docosahexaenoic acid (DHA), could be identified in the cytosol of rat and bovine retinas under in vitro and in vivo conditions. METHODS In vitro, cytosol fractions from normal bovine and rat retinas were delipidated and incubated with [14C]-DHA with or without a number of competing fatty acids. After crosslinking bound FA to BP, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioactivity determined in each fraction. For in vivo experiments, rats received [14C]-DHA by gavage. At selected periods after ingestion, retinas were collected and cytosolic fractions were prepared from each. These were crosslinked and subjected to SDS-PAGE, and radioactivity was determined in each fraction. RESULTS In vitro, several peaks of radioactivity (approximately 13, 20, 32, 43-45, 50, 63, and 94-105 kd) were found that exhibited [14C]-DHA binding. Relative specificity of binding was assessed by blocking of the [14C]-DHA binding with unlabeled DHA and by competition experiments with other FAs. After in vivo ingestion of [14C]-DHA, a large peak of radioactivity was observed at 43 kd by 4 hours. At 6 hours, this peak decreased and, after 24 hours, it approached baseline. CONCLUSIONS These findings demonstrate that there is a discrete grouping of proteins in retinal cytosol capable of binding DHA and other FA. Although the identities of these proteins have yet to be determined, the group may include a member of the 12- to 15-kd group of small fatty acid binding proteins (FABP). In particular, a unique 43-kd binding peak could play a major role in the uptake, binding, or both of DHA by the retina in vivo.