Triacylglycerols (TAG) were purified from the storage lipids extracted from the seeds of several conifer species (Taxus baccata, Larix decidua, Sciadopytis verticillata, and Juniperus communis), each species belonging to one of the four families Taxaceae, Pinaceae, Taxodiaceae, and Cupressaceae, respectively. Each species was characterized by a high content of 5,9-18:2, 5,9,12-18:3, 5,11,14-20:3, or 5,11,14,17-20:4 acids, respectively. TAC were partially deacylated with ethyl magnesium bromide, and the resulting 1,2-, 2,3-diacylglycerols (DAG), and 2-monoacylglycerols (MAC) were purified by thin-layer chromatography. 1,2- and 2,3-DAG were further fractionated by chiral column high-performance liquid chromatography of the 3,5-dinitrophenylurethane derivatives. Alternately, TAG were subjected to porcine pancreatic lipase, and the resulting 2-MAG were purified for further analysis. Gas-liquid chromatography of fatty acid methyl esters prepared from the separated DAC and MAC, coupled with appropriate calculations, indicated that the Delta 5-olefinic acids, irrespective of the species, chainlengths and number of ethylenic bonds, were considerably enriched in the sn-3 position of TAC where they accounted for ca. 35 to 74 mole% of fatty acids esterified to this position (depending on the initial level of total Delta 5-olefinic acids in TAG), which corresponded to 79-94% of Delta 5-olefinic acids esterified to the three positions. On the other hand, Delta 5-olefinic acids were less than 10% in the sn-2 position and less than 6% in the sn-1 position of TAG. This specific enrichment of Delta 5-olefinic acids in the sn-3 position thus appears to be a general characteristic of conifer seed TAG. These results were extended to TAG from the seeds of two pine species (Pinus koraiensis and P. pinaster) that are rich in Delta 5-olefinic acids and available commercially on a ton-scale.