The positional distribution of various Delta 5-acids in the seed triacylglycerols from several conifer species has been established after partial chemical degradation with Grignard reagent. The species studied were representative of four conifer families and were specially selected for their particularly high Delta 5-acid contents. These species were Taxus baccata (Taxaceae; 5,9-18:2 acid, 11.9%), Larix decidua (Pinaceae; 5,9,12-18:3 acid, 28.5%), Sciadopytis verticillata (Taxodiaceae; 5,11,14-20:3 acid, 16.7%), and Juniperus communis (Cupressaceae; 5,11,14,17-20:4 acid, 19.8%). Calculations from the fatty acid compositions of triacylglycerols and of the mixture of 1,2- and 2,3-diacylglycerols generated by the Grignard reagent indicated that, for the four species, there was a considerable enrichment of Delta 5-acids (generally more than ten times) in the 1,3-positions as compared to the 2-position, where Delta 5-acids represented always less than 2% of total fatty acids esterified to triacylglycerols. This distribution was practically independent from the species (four families studied), the chainlength (18 or 20 carbon atoms), and the number of ethylenic bonds (two to four) in the Delta 5-acids. Similar distributions were established for triacylglycerols from the seeds of three pine species that are available on a ton-scale: Pinus pines, P. koraiensis, and P. pinaster. These observations confirm and extend previous studies conducted with other conifer species by similar techniques or by C-13-nuclear magnetic resonance spectroscopy. Consequently, the almost exclusive location of Delta 5-acids in the external positions of triacylglycerols is now well established and appears to be a general feature of conifer seed oils.