In the accompanying paper, we demonstrated that Chlorella kessleri uses prokaryotic and eukaryotic pathways to synthesize sn-1-C18-sn-2-C16 (C18/C16, prokaryotic lipids) and sn-1-C18-sn-2-C18 (C18/C18, eukaryotic lipids) species, respectively, in chloroplast lipids such as monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG). In this study, to examine the effect of CO2 on lipid metabolism, we compared the fatty acid distributions at the sn-1 and sn-2 positions of each major lipid, i.e. MGDG, DGDG, phosphatidylcholine (PC), and phosphatidylethanolamine (PE), and the patterns of incorporation of [14C]acetate into fatty acids and lipids in vivo between cells of C. kessleri grown under ordinary air (low-CO2 cells) and ones grown under CO2-enriched air (high-CO2 cells). Low-CO2 cells, as compared with high-CO2 cells, showed elevated contents of 18:3(9,12,15), especially at both the sn-1 and sn-2 positions of MGDG and DGDG, and also at the sn-2 position of PC and PE. When the cells were labeled with [14C]acetate, slower rates of 18:3 synthesis in the respective major lipids with lower incorporation of 14C into total membrane lipids were observed in low-CO2 cells than in high-CO2 cells. These results thus indicate that the higher unsaturation levels in low-CO2 cells are at least partially due to repressed fatty acid synthesis, which promotes the desaturation of pre-existing fatty acids, rather than to up-regulation of desaturation activity. It was also noted that, in both MGDG and DGDG, the contents of eukaryotic lipids were higher at the expense of prokaryotic lipids in low-CO2 cells than in high-CO2 cells, suggesting relatively greater metabolic flow in the eukaryotic pathway compared to the prokaryotic pathway for galactolipid synthesis in low-CO2 cells. We propose that, together with the repression of fatty acid synthesis, the increased synthesis of C18/C18 species of galactolipids, which are suitable substrates for chloroplast desaturation, through the eukaryotic pathway, contributes to the higher contents of 18:3 in low-CO2 cells than in high-CO2 cells.