Abstract Estradiol-binding macromolecules in fetoneonatal rat brain cytosol and serum were compared by immunochemical techniques. When tested by a double diffusion procedure, both cytosol and serum formed precipitin lines with rabbit antiserum specific for perinatal rat serum proteins. These lines fused completely, indicating, within the limits of detection of this particular antiserum, the presence of identical antigenic determinants in the brain and serum. Prior removal of immunoprecipitable material from cytosol or serum, after incubation with the specific antiserum, prevented formation of such precipitin lines. This procedure similarly prevented specific estradiol-binding to macro-molecules. It was therefore concluded that the specifically perinatal, antigenically similar, components in rat brain cytosol and serum (possibly representing alphafetoprotein) are responsible for the estra-diol-binding activity in these two tissue compartments. Measurements of heme concentrations indicated that the alphafetoprotein-like material in the cytosol does not reflect blood contamination, but rather a separate population of similar or identical molecules.