Maternal riboflavin-binding protein (RfBP) mediates the deposition of riboflavin in avian eggs. A strain of Single-Comb White Leghorn chicken, genetically unable to synthesize RfBP, produces eggs deficient in riboflavin. Embryos in these eggs die on or about 13 of incubation, with severe hypoglycemia and impaired fatty acid oxidation. Injection of free riboflavin prior to incubation allows these embryos to survive and hatch normally. The egg whites of eggs from the RfBP-deficient strain were injected with different relative amounts of riboflavin and RfBP to determine their effects on the developing embryo. Injection of protein-bound riboflavin resulted in a substantial number of the embryos (27%) surviving until the end of the experiment (d 19) with an average weight of 13.7 g, demonstrating that some embryos were able to use the bound riboflavin. In control groups injected with riboflavin alone, 10 of 17 embryos survived until d 19 with an average weight 18.5 g, whereas those injected with saline died between d 10 and 13 with an average weight of 3.8 g. A fourth group, injected with apo-RfBP, produced embryos with an average weight of 0.24 g. This early death suggests that the protein scavenges the small amount of riboflavin normally available to the early embryo. Further studies showed that titration of RfBP-binding sites with exogenous riboflavin lessens this effect. These studies show that although riboflavin-binding protein is essential for the deposition of riboflavin in eggs, its presence in excess can diminish the availability of riboflavin to the chicken embryo.