Biosynthesis of alpha-linolenate was investigated in developing embryos of the high-linolenic (45%) linseed cv. Glenelg, two mutant lines (M1589 and M1722) having reduced linolenic acid content (30%), and a very low linolenic (2%) genotype (Zero) obtained by recombination of the M1589 and M1722 mutations. Glenelg embryos showed an exponential rate of linolenate synthesis that paralleled their exponential pattern of triacylglycerol accumulation. The Zero line, although showing a pattern of triacylglycerol accumulation similar to that of Glenelg, accumulated linolenate at only a very low and constant rate throughout embryo development. An NADH- and O2-dependent decrease in oleate and increase in linolenate content of phosphatidylcholine was observed in dilute homogenates prepared from Glenelg embryos at 21 days after flowering, indicating active oleoyl- and linoleoyl-phosphatidylcholine desaturases in these preparations. While oleate decreased similarly in both sn positions of phosphatidylcholine, the increase in linolenate was confined mostly to the sn-2 position. Homogenates prepared from the mutant lines showed decreases in oleoyl-phosphatidylcholine similar to those of the wild-type Glenelg, whereas the increase in linolenoyl-phosphatidylcholine was substantially lower in M1589 and M1722 and barely detected in Zero. In vivo labeling experiments with detached embryos at 17 days after flowering, as well as analysis of endogenous linolenate content in various lipids, indicated that only delta 15-phospholipid desaturases, and not delta 15-galactolipid desaturases, were affected by the mutations. Embryos from M1722 had amounts of both radioactive and endogenous linolenate at position sn-1 of phosphatidylcholine that were close to those of the wild-type embryos, whereas M1589 had only 30 and 50% of these levels, respectively. The regulation of linolenic acid content in oilseeds is discussed on the basis of the results obtained.