We have compared the molecular species composition of the glycerolipids of rat liver and rat plasma very low density lipoproteins (VLDL). There were differences in the stereospecific distribution of the fatty acids in the triacylglycerols (TG) of the liver and of VLDL. While chiral and reversed phase chromatography with mass spectrometry (LC/MS) revealed great similarities in positional distribution and molecular association of the fatty acids between the sn-1,2-diacylglycerol (DG) moieties of the VLDL and liver TG, the corresponding sn-2,3-DG were distinctly different. The free hepatic sn-1,2-DG and the sn-1,2-DG moiety contained within hepatic phosphatidic acid showed a maximum 60% homology to the sn-1,2-DG contained within the TG of the liver and of VLDL. By contrast, the smaller pool of hepatic free sn-2,3-DG was nearly identical to the sn-2,3-DG moiety contained in the TG of the liver. These differences between hepatic and VLDL TG indicate that direct transfer of hepatic triacylglycerols is not a major mechanism of VLDL TG formation. On the other hand, the results suggest that stored hepatic TG are largely hydrolyzed to sn-1,2-DG and then reesterified to TG before being secreted as VLDL TG. Although an involvement of 2-monoacylglycerol pathway could not be excluded, it probably plays a minor role in VLDL TG formation. Our data suggest that a minimum of 60% of the VLDL TG could have been derived via hydrolysis to DG and reesterification, and a maximum of 40% could have originated via the conventional phosphatidic acid pathway.