- In the course of the study of the biosynthesis of the fatty acid eicosapentaenoic acid (EPA) in the microalga Porphyridium cruentum, cells were pulse-labeled with various radiolabeled fatty acid precursors. Our data show that the major end products of the biosynthesis are EPA-containing galactolipids of a eukaryotic and prokaryotic nature. The prokaryotic molecular species contain EPA and arachidonic acid at the sn-1 position and C16 fatty acids, mainly 16:0, at the sn-2 positions, whereas in the eukaryotic species both positions are occupied by EPA or arachidonic acid. However, we suggest that both the eukaryotic and prokaryotic molecular species are formed in two pathways, [omega]6 and [omega]3, which involve cytoplasmic and chloroplastic lipids. In the [omega]6 pathway, cytoplasmic 18:2-phosphatidylcholine (PC) is converted to 20:4[omega]6-PC by a sequence that includes a [delta]6 desaturase, an elongation step, and a [delta]5 desaturase. In the minor [omega]3 pathway, 18:2-PC is presumably desaturated to 18:3[omega]3, which is sequentially converted by the enzymatic sequence of the [omega]6 pathway to 20:5[omega]3-PC. The products of both pathways are exported, as their diacylglycerol moieties, to the chloroplast to be galactosylated into their respective monogalactosyldiacylglycerol molecular species. The 20:4[omega]6 in both eukaryotic and prokaryotic monogalactosyldiacylglycerol can be further desaturated to EPA by a chloroplastic [delta]17 ([omega]3) desaturase.