Previous studies have shown several similarities between an estrogen-regulated heat shock protein of 24,000-28,000 daltons (hsp27), and a cytoplasmic estrogen receptor-associated protein of 27,000-29,000 daltons (p29). These proteins have been studied by monoclonal antibodies generated in different laboratories. In the present report we have performed immunocytochemical and immunoblot studies to explore if the monoclonal antibodies against hsp27 (C11) and against p29 (ER-D5) may be identifying the same protein. Breast and endometrial carcinomas and normal endometrial samples were examined by immunocytochemistry (in mirror sections and by double-immunostaining). Identical hsp27 and p29 immunostaining intensity, distribution, and percentage of stained cells was demonstrated by immunocytochemistry. The antigens examined by the two antibodies appeared in the same cells. Cytosols from tumors analyzed by the Western blot technique revealed that the C11 and the ER-D5 antibodies recognized bands with identical electrophoretic mobility. Immunoprecipitation studies with one antibody, C11, followed by Western blot showed that the precipitate was reactive with both antibodies. Identical C11 and ER-D5 reacting spots were observed after blotting proteins separated by high resolution two-dimensional gel electrophoresis. In addition, p29 protein was induced by heat shock in the estrogen receptor negative MDA-MB-231 human breast tumor cell line. These results strongly suggest that the two proteins under investigation are identical.