Microsomes prepared from developing safflower seeds rapidly desaturated added [14C]oleoyl-CoA in the presence of NADH. The distribution of [14C]oleate and [14C]linoleate between different lipid classes revealed that phosphatidylcholine was labelled with [14C]linoleate before any other lipid class investigated. Considerable desaturation continued after the disappearance of [14C]oleoyl-CoA in the reaction mixture. No [14C]linoleoyl-CoA could be detected. Incubation in the absence of added NADH caused a rapid disappearance of [14C]oleoyl-CoA by incorporation into triacylglycerols and phosphatidylcholine and also by release as [14C]oleic acid. Upon subsequent addition of NADH, [14C]linoleate was formed to the same extent as when NADH was present at the onset of the incubation. These data are contradictory to the previously assumed pathway for linoleic acid biosynthesis in developing safflower seeds, claiming oleoyl-CoA as the direct substrate and linoleoyl-CoA as the primary product. However, our data corroborate published results on Chlorella, Candida, Torulopsis and on developing pea leaves, which strongly suggest that oleoyl-CoA is first incorporated into phosphatidylcholine and then desaturated to linoleoyl-phosphatidylcholine.