The purpose of our study was to show the feasibility of accurately investigating the factors likely to control cell proliferation of normal human mammary epithelial (HME) cells, using a scanning cytometric method. The methodology was previously developed with the SAMBA 200 cell image processor to characterize in situ the cell-cycle phases of HME cells. Since various compartments constitute the mammary epithelium, cells obtained after reduction mammoplasty were cultured in a medium with a low calcium content (0.06 mM) to provide proliferating normal HME cells while maintaining their differentiation characteristics. Estradiol and EGF requirements for cell-cycle phase progression of these cells were examined. Then, we showed that 2 normal HME cell cultures displaying different phenotypic characteristics may differently progress through the cell cycle under the same hormonally defined conditions. Cell-cycle progression of the epithelial cells presenting luminal phenotype was induced only by sequential stimulation/pretreatment with estradiol followed by EGF treatment; this progression was enhanced when estradiol was maintained during EGF treatment. This definite order demonstrated that estradiol could have a permissive effect on EGF mitogenic activity. In contrast, epithelial cells likely to be localized in the basal position in the mammary gland showed the same proliferating activity whatever the estradiol and EGF treatment. These cells progressed profusely in cell cycle, independent of exogenous estradiol and EGF contribution, but they remained sensitive to estradiol regarding EGF-receptor detection. Our results suggest autonomous proliferation of these cells through an autocrine pathway, in the absence of negative regulators.