Investigations of storage lipid synthesis in developing flaxseed (Linum usitatissimum) provide useful information for designing strategies to enhance the oil content and nutritional value of this crop. Lipid content and changes in the FA composition during seed development were examined in two cultivars of flax (AC Emerson and Vimy). The oil content on a dry weight basis increased steadily until about 20 d after flowering (DAF). The proportion of alpha-linolenic acid (alpha-18:3, 18:3cisDelta9,12,15) in TAG increased during seed development in both cultivars while the proportions of linoleic acid (18:2cisDelta9,12) and saturated FA decreased. The developmental and substrate specificity characteristics of microsomal DAG acyltransferase (DGAT, EC 2.3.1.20) and lysophosphatidic acid acyltransferase (LPAAT, EC 2.3.1.51) were examined using cultivar AC Emerson. The maximal acyltransferase specific activities occurred in the range of 8-14 DAF, during rapid lipid accumulation on a per seed basis. Acyl-CoA of EPA (20:5cisDelta5,8,11,14,17) or DHA (22:6 cis4,7,10,13,16,19) were included in the specificity studies. DGAT displayed enhanced specificity for alpha-18:3-CoA, whereas the preferred substrate of [PAAT was 18:2-CoA. Both enzymes could use EPA- or DHA-CoA to varying extents. Developing flax embryos were able to take up and incorporate these nutritional FA into TAG and other intermediates in the TAG-formation pathway. This study suggests that if the appropriate acyl-CoA-dependent desaturation/elongation pathways are introduced and efficiently expressed in flax, this may lead to the conversion of alpha-18:3-CoA into EPA-CoA, thereby providing an activated substrate for TAG formation.