Estradiol-binding sites, as assayed by exchange with radiolabeled steroid, become associated with the nuclear matrix of estrogen-responsive tissues after treatment with estrogen in vivo. Using monoclonal estrogen receptor antibodies, we have now obtained direct evidence that these matrix-associated estradiol-binding sites are estrogen receptor proteins similar to those found in the cytosol before estrogen treatment. Proteins of the liver nuclear matrix from untreated or ethinyl estradiol-treated female rats were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose paper, and probed with the monoclonal estrogen receptor antibody H222Sp gamma. A single prominent immunoreactive 67,000 mol wt band, indicating the presence of estrogen receptors, was found in the liver nuclear matrix of estrogen-treated animals. This band was detectable, but of much lower intensity, in the liver nuclear matrix of untreated animals. Liver cytosol estrogen receptor from untreated rats also migrated as a 67,000 mol wt band. These immunoreactivity data corroborated data obtained by [3H]estradiol-binding assays. Scatchard analysis of specific high affinity [3H]estradiol-binding sites showed high levels of these sites in the liver nuclear matrix of estrogen-treated rats and low levels in untreated rats. Therefore, both direct and indirect methods of receptor identification demonstrate the specific association of estrogen receptors with the nuclear matrix after estrogen treatment in vivo.