The glycerol portion of lipids may be derived biosynthetically by reduction of dihydroxyacetone phosphate to glycerolphosphate, and then be acylated with fatty acids or, alternatively, dihydroxyacetone phosphate may first be acylated and then reduced to 1-acyl sn glcerol-3-phosphate. Since the former pathway utilizes NADH for reduction of the C-2 carbonyl, while the latter requires NADPH, we were able to compare the relative participation of the two pathways for phospholipid synthesis by measuring the incorporation of radioactivity from tritiumlabeled NADH and NADPH into C-2 of lipid glycerol. The acyl-dihydroxyacetone phosphate pathway plays a significant role in glycerolipid synthesis in mouse liver homogenates and a clearly dominant one in Ehrlich ascites tumor cell homogenates. This finding is related to a reported lack of glycerol-3-phosphate dehydrogenase in tumor cells and to their high glycerol ether lipid content.