The cytoplasmic estrogen binding proteins (EBP) of mammary glands were characterized using sucrose gradient centrifugation and Sephadex G-200 gel chromatography. The EBP obtained with ammonium sulfate precipitation tends to aggregate in both low and high ionic strength buffers in the presence of EDTA. If the ammonium sulfate precipitate is redissolved in buffers without EDTA, the EBP sediments as a discrete 8S molecule under low ionic conditions and as a 4S EBP with 0.4 M KCl. In addition to EDTA, the aggregation of EBP is caused by various structurally different metal chelators and this effect can be reversed by the addition of Zn ++ or Mn ++. It is proposed that the cytoplasmic EBP of mammary glands may contain a bound metal ion which may be crucial for the confirmation of the protein favorable to the binding of the steroid.