Administration of estrogen to roosters induces the synthesis of egg yolk phosphoproteins in the liver. We have demonstrated that these proteins are synthesized in the form of a large precursor, vitellogenin, and that vitellogenin is the only phosphoprotein found in the plasma of the estrogen-treated rooster. Vitellogenin is cleaved to form the egg yolk phosphoproteins, lipovitellin, and phosvitin. We have purified vitellogenin to hemogeneity by two methods: chromatography on diethylaminoethyl-52-cellulose and affinity chromatography on an antibody-Sepharose column. Antibodies were elicited in rabbits and sheep by immunization with vitellogenin and lipovitellin, and these antibodies were purified by affinity chromatography on antigen-Sepharose columns. We found that phosvitin was not immunogenic in its native form or in any of the large variety of modified forms we have tested. We have determined the molecular weights of native and denatured vitellogenin and have examined the immunological relationship between vitellogenin and lipovitellin. On the basis of these studies, together with data from phosphate analyses, we suggest that avian vitellogenin is composed of two polypeptides, each of which has a molecular weight of approximately 240,000 and contains within it lipovitellin and two phosvitins.