Estrogen receptor (ER) in human breast cancer tissues was demonstrated in paraffin sections as well as in frozen sections by immunoperoxidase methods using monoclonal antibody (H222) against ER. The avidin-biotin-peroxidase complex method was used for the paraffin sections fixed in cold buffered formalin, and the peroxidase-antiperoxidase method was used for the fixed frozen sections. The results were compared with the ER content in the respective tumor tissue determined by dextran-coated charcoal assay. The specific staining for ER was located exclusively in the nuclei of cancer cells in both paraffin and frozen sections. Differences in the intensity and distribution of nuclear staining within a section were often observed, suggesting heterogeneity of the ER content of individual breast cancer cells. In 24 breast cancer tissues studied simultaneously by both paraffin and frozen section methods, 21 (88%) showed similar evaluation of the presence of ER. The results of immunocytochemical staining agreed with those of the dextran-coated charcoal assay in 89 (82%) of the 109 paraffin-sectioned tumor tissues and in 24 (86%) of the 28 frozen-sectioned tissues, indicating that ER can be demonstrated immunocytochemically by use of paraffin as well as frozen sections.