Docosahexaenoic acid (22:6n-3) can be synthesized in the liver and/or brain from alpha-linolenic acid (18:3n-3) and is required in large amounts in structural membranes of developing brain and retina. The adequacy and efficacy of formulas containing 18:3n-3 and/or fish oil in providing 22:6n-3 for deposition was investigated in piglets fed formula from birth to 15 days. The test formulas contained high (HL) or low (LL) 18:3n-3 (3.9 or 0.7% of the total formula fatty acids, respectively), or low 18:3n-3 plus fish oil (LL+FO) to provide C20 and C22 n-3 polyunsaturated fatty acids (0.8% of total fatty acids). Fatty acid analyses of synaptic plasma membrane and retina ethanolamine phospholipids (EPL), which are especially enriched in 22:6n-3, were compared to those of 15-day-old piglets fed sow milk (SM). Feeding LL resulted in lower 22:6n-3 in synaptic plasma membrane. Fatty acid levels in HL and LL+FO piglets were equivalent to SM, with the exception of lower 22:5n-3 in the synaptic plasma membrane of LL+FO and in the retina of HL and LL+FO-fed piglets. Levels of 22:4n-6 were also lower in the retina of the LL+FO group. The results suggest formula 18:3n-3 is at least 24% as effective as C20 and C22 n-3 fatty acids as a source of membrane 22:6n-3. This study shows dietary 18:3n-3, as the only n-3 fatty acid, can support deposition of comparable percentage of 22:6n-3 to natural milk. Fish oil also supported tissue levels of 22:6n-3 similar to natural milk; however, lower 22:4n-6 may indicate possible inhibitory effects on n-6 metabolism.