Primary cultures of bovine adrenal medullary cells synthesize and secrete a high-molecular-weight protein into the culture medium. The protein was purified from the serum-free medium of cultured cells and was identified as α2-macroglobulin by gel electrophoresis, sedimentation velocity, electron microscopy, immunoprecipitation, immunodiffusion, and autoradiography. Antisera directed against the protein were prepared and used to determine the cell types that synthesize the protein. Immunohistofluorescence studies show that adrenal cortical cells present in the adrenal medullary cell cultures reacted with the antisera to the protein purified from the medium, but adrenal medullary chromaffin cells did not. Cell cultures prepared from bovine adrenal cortex also synthesize and secrete α2-macroglobulin and react with the antisera.