The objective was to determine the contributions of glucose to glycogen synthesis and glycerol to glycogen, glucose, and nonessential AA (NEAA) synthesis on embryonic day (e) 14/15 and e19/20. Chicken embryos from small (56.6 ± 0.88 g) and large eggs (71.7 ± 1.09 g) were repeatedly dosed with either [(13)C(3)]glycerol (14 mg/d for 4 d) or [(13)C(6)]glucose (15 mg/d for 3 d) into the chorio-allantoic fluid before blood and tissue collection. (13)C-Mass isotopomer enrichments in blood glucose, liver, and muscle glycogen, and blood and tissue NEAA were analyzed by mass spectrometry. Glucose metabolism did not differ between small- and large-egg embryos. Although glucose entry was 60% less for e20 compared with e15 embryos, e20 embryos conserved glucose more efficiently as a result of 2- to 3-fold greater (P < 0.001) rates of glucose carbon recycling. Importantly, the direct contribution of glucose to liver glycogen synthesis was minimal on e15, and on e20 direct incorporation of glucose into liver glycogen was only 17%. By comparison, [(13)C(3)]glycerol dosing led to the appearance of [M + 1], [M + 2], and [M + 3] isotopomers in blood glucose and in liver and muscle glycogen on e14 and e19. Here, the (13)C-isotopomer enrichments in blood glucose were ~2-fold greater (P < 0.05) in small- than in large-egg embryos on e14 and e19. Furthermore, [(13)C(3)]glycerol dosing led to substantial labeling of [M + 1], [M + 2], and [M + 3] isotopomers of alanine, aspartate, and glutamate in blood and in tissues where (13)C enrichments were greater (P < 0.05) in liver of small-egg embryos. In summary, this study provides unequivocal evidence that glycerol is a precursor for glucose and NEAA synthesis. Furthermore, glycerol, but not egg-derived glucose, is a major substrate for synthesis of liver and muscle glycogen and is an important anaplerotic substrate for the tricarboxylic acid cycle of embryos during later development.