Splenic lymphocytes from a Lewis rat, immunized with purified estradiol-receptor complex of calf uterine nuclei, were fused with cells of three different mouse myeloma lines (P3-X63-Ag8, P3-NSI/I-Ag4-1, and Sp2/0-Ag14) to yield hybridoma cultures, 9% of which produced antibodies to the receptor protein (estrophilin). When cloned by limiting dilution, approximately 70% of the viable cultures secreted antiestrophilin antibody. When expanded in suspension culture, three clones derived from Sp2/0-Ag14 were found to secrete rat IgG (gamma 2a class), whereas seven other clones (from all three myeloma lines) secreted IgM. Monoclonal IgG shows comparable affinity for nuclear and extranuclear receptors, whereas IgM reacts preferentially with the nuclear form. Both classes of antibody react with unoccupied as well as with occupied receptor and do not interfere with its ability to bind to estradiol. By growing IgG-secreting clones in the presence of [35S]methionine, radiolabeled monoclonal antiestrophilin has been prepared. Unlike antiestrophilin antibody previously generated in the rabbit or the goat, which crossreacts with estrogen receptors from every animal species tested, antibodies produced by the Lewis rat and by hybridomas derived from its spleen cells react specifically with estrophilin from calf tissues. These monoclonal antibodies provide reagents for the application of immunochemical techniques to study estrogen receptors in calf target tissues.