An estrogen-regulated cytoplasmic protein has been purified from MCF-7 human breast cancer cells with anion exchange and monoclonal antibody affinity chromatographies. The purified protein has a monomeric mol. wt of 28,000 and isoelectric species with pI's between 5.9 and 6.0. Amino acid analysis indicates the protein is acidic, is probably hydrophilic, and contains unusually low amounts of methionine and half cystine. The monoclonal antibodies are of the IgG1 subclass, exhibit a high affinity for the 28K protein, and recognize an antigenic site that is stable to sodium dodecyl sulfate (SDS) gel electrophoresis and electrophoretic transfer techniques. These monoclonal antibodies have been used to detect 28K in certain human breast tumors.