A method is described for measuring the rate of phenotypic variability in normal and neoplastic breast epithelial cells. Three groups of normal human mammary epithelial cells were studied, two derived from reduction mammoplasties and one derived from the normal breast tissue of a patient with fibroadenoma. The breast carcinoma cells were all cell lines, four (MCF-7, SKBR-3, MDA-MB-157, and T47D) derived from pleural effusions of patients with breast cancer, and one (BT-20) derived from a primary breast tumor. The heterogeneity and variability in expression of a cell surface glycoprotein with apparent molecular weight of 400,000 were studied at the single-cell level with immunoperoxidase techniques using a specific monoclonal antibody, BLMRL-HMFG-Mc5, to a nonpenetrating glycoprotein. The rate of appearance of quantitative variants in expression of this specific surface antigen (rate of phenotypic variability) was determined in clonal colonies and was found to be severalfold higher in all five breast carcinoma cell lines (mean, 2.23 X 10(-2)/cell/generation) than in the normal breast epithelial cells (mean, 0.36 X 10(-2)/cell/generation). In addition, a considerable quantitative variation in expression of this surface antigen was demonstrated among the cells of each population in both normal and neoplastic breast cells which spread over an 8- to 10-fold range. Furthermore, the quantitative distribution among single cells was not random, for the cells tended to cluster around values that fit a geometric series.