The percentage of saturated cholesteryl esters (CEs) synthesized by human LCAT is several times higher than expected from the sn -2 acyl composition of plasma phosphatidylcholine (PC), whereas the synthesis of 20:4 CE and 22:6 CE is much lower than expected. To explain these discrepancies, we proposed that LCAT transfers some saturated fatty acids from the sn -1 position of PC species that contain 20:4 or 22:6 at sn -2. The present studies provide in vivo evidence for this hypothesis. We determined the composition and synthesis of CE species in plasma of volunteers before and after a 6 week dietary supplementation with docosahexaenoic acid (22:6; DHA). In addition to an increase in the DHA content of all plasma lipids, there was a significant ( ؉ 12%; P Ͻ 0.005) increase of 16:0 CE, although there was no increase in 16:0 at sn -2 of PC. The increase of DHA in CE was much lower than its increase at sn -2 of PC. Most of the cholesteryl esters (CEs) in the lipoproteins of human plasma are derived from the action of LCAT (1). Although this enzyme has been shown to be specific for the sn-2 position of phosphatidylcholine (PC), the composition of the CEs in human plasma does not match that of the sn-2 acyl group (1-4). For example, although 16:0 constitutes only ف 2-3% of the PC sn-2 acyl groups, it accounts for 10-12% of plasma CE. On the other hand, although 20:4 constitutes ف 16% of the sn-2 acyl groups of PC, only ف 5% of the plasma CE is 20:4. Similarly, the concentration of 22:6 at sn-2 of PC ( ف 5%) is much higher than that in CE ( ف 0.4%). Although these discrepancies have been attributed to the preference of the enzyme for the PC substrates containing 16:0 at sn-2 (5), in vitro studies with synthetic substrates and isolated enzyme did not support this mechanism (6). Our studies on the use of PC species by LCAT in native plasma also showed that 16:0-16:0 PC, the major PC with 16:0 at sn -2, is not preferred over "average" PC in plasma. Based on our studies with synthetic PC substrates and isolated LCAT (7, 8), we proposed that human LCAT transfers significant amounts of sn-1 acyl group from the PC species containing 20:4 or 22:6 at the sn -2 position. This mechanism not only explains the synthesis of higher than expected percentages of saturated CE species but also accounts for the formation of lower than expected percentages of 20:4 and 22:6 CE. This alteration in positional specificity appears to be a function of the architecture of the enzyme's active site, because rat and mouse enzyme are not substantially affected by the presence of sn-2-20:4 PC (8) and because the human LCAT behaves like the mouse enzyme if its active site domain is replaced by the corresponding domain from mouse enzyme (4, 9).Although we have provided multiple lines of evidence for the altered positional specificity of human LCAT in vitro, there is no direct evidence that this occurs in vivo. Previous studies of the effects of fish oil feeding in humans (10) and nonhuman primates (11,12) reported an increase in the ap...