The objective of this study was to determine if 20:5n-3 or 22:6n-3 is the primary precursor of very-long-chain fatty acids (VLCFAs; C24-C36) synthesized in retina. Rats were fed semisynthetic, nutritionally complete diet containing 20% (w/w) fat with 3% (w/w) of 22:6n-3. After 6 weeks feeding, the vitreal fluid of each eye was injected with [3H]20:5n-3 or [3H]22:6n-3. Rats were then maintained under constant light (330 lux) or dark conditions for 48 hr. After 48 hr in vivo metabolism, the amount of label present in individual fatty acids was determined in major phospholipids in retina. For [3H]22:6n-3, 90% of total incorporation remained in 22:6n-3, whereas for [3H]20:5n-3 the label was actively incorporated into pentaenoic and hexaenoic VLCFAs up to 34 carbon chain length. 22:5n-3 derived from [3H]20:5n-3 was among the most highly labeled fatty acids. These observations suggest that 22:6n-3 is incorporated directly into retinal phospholipids without further metabolism, whereas 20:5n-3 and 22:5n-3 are metabolically active precursors for synthesis of VLCFAs.