We have analyzed the frequency and distribution of cells expressing estrogen receptor (ER) in cryosections of normal human breast tissue using quantitative microspectrophotometry and the estrogen receptor immunocytochemical assay. We found that the human mammary gland contained a small but distinct population of ER-positive cells, comprising approximately 7% of the total epithelial cell population from all biopsies. Stromal cells were found to be ER negative. The ER-positive cells were distributed as scattered single cells, with the highest frequency and intensity of measured staining in the lobules as compared to the interlobular ducts. Moreover, on the average, 87% of the ER-positive cells were luminal epithelial cells or occupied an intermediate position in the duct wall. The intermediate cells were found not to express basal cell phenotype as determined by combined immunocytochemistry to ER and “common acute lymphoblastic leukemia antigen” selectively decorating myoepithelial cells (B. A. Gusterson et al., J. Natl. Cancer Inst., 77: 343–349, 1986).