Although the literature included relatively little evidence regarding the role of different cholesteryl esters. Determining the plasma profile of unesterified fatty acids is normally problematic due to the direct influence of diet on plasma fatty acid content. Using a single diet for both WT and fat-1 animals made it possible for plasma content analysis to be used as a reliable indicator of endogenous PUFA metabolism. Notably, the diet does not contain longer chain PUFAs and any circulating lipids longer than 18 carbons must come from endogenous metabolism of shorter PUFAs. Our study highlighted a significant difference in the omega-6/omega-3 ratio between unesterified plasma lipids and other previously reported tissues. In particular, our results showed a marked increase for unesterified EPA compared to unesterified DHA in the plasma of fat-1 mice, which could be explained, in part, by a retroconversion of DHA to EPA. The concomitant elevation of the DHA-containing phospholipids, however, suggests that EPA might be a preferred substrate for the hydrolytic activity of phospholipase A2 in plasma, rather than DHA. These observations point to a differential metabolism for EPA and DHA, which could explain the diverse physiological effects previously reported for these two omega-3 PUFAs. On the other hand, DHA-containing phospholipids and lysophospholipids may play roles in the cellular membrane properties and in the transport of DHA to other tissues. Lysophospholipids containing DHA do, in fact, appear to be best suited as carriers of this essential fatty acid to eye and brain tissues, where it modulates the membrane fluidity of synaptic vesicles and displays neuroprotective properties. Notably, our untargeted analysis highlights a wide increase in other lysophospholipid species, which comports with a previous report showing that dietary supplementation with omega-3s can dynamically regulate plasma lysoPC. The untargeted lipidomic analysis also showed a significant decrease in triacylglycerols and cholesterol. Such a decrease is in accordance with the fact that the fat-1 phenotype is resistant to metabolic syndrome, obesity, and liver steatosis. Significantly, the unexpected decrease in cholesterol-sulfate in the plasma of fat-1 mice may also be linked with some of the protective effect of omega-3s. Cholesterol sulfate is present in lipoproteins, and it has been found in atherosclerotic lesions of the human aorta, where it plays a role in platelet adhesion, possibly determining the prothrombotic potential of atherosclerotic lesions. Cholesterol sulfate is also found to be particularly enriched in DHA-rich cellular membranes, where it seems to modulate the lipid raft formation. One might reasonably speculate that the observed decrease in the circulating levels of cholesterol sulfate might indicate the effect of possible sequestration caused by the DHA-rich cellular membranes in fat-1 mice.