The mouse mammary gland undergoes rapid proliferation during puberty, then cyclical proliferation and involution during adulthood within a 5-day estrous cycle. Although proliferation of mammary epithelial cells is directed by elevated serum levels of estrogen acting via the estrogen receptor (ER), the ER status of the proliferating cells remains unknown. We examined the ER expression of proliferating epithelial cell types during pubertal development and normal adult growth using simultaneous immunohistochemistry for ER and tritiated thymidine (3H-Tdr) autoradiography. These studies demonstrate that during pubertal growth (4-6 weeks) ER-negative cells comprise more than 50% of the epithelial cell populations in the terminal end buds (TEBs) and ducts. Furthermore, the majority of proliferating cells in both TEBs and ducts are ER-negative. These findings indicate that proliferation of cells within both the TEBs and the mammary ducts contribute to pubertal growth of the mammary gland and that the greater proportion of dividing cells are ER-negative. Similar patterns of cell growth were observed in the normal estrous cycle when the majority of dividing cells were ER-negative during both pro-estrous and estrous. Intensive labelling of cells with 3H-Tdr was used to identify long-lived mammary epithelial cells which retained 3H-Tdr 2 weeks following labelling (i.e., following 3 estrous cycles). Of the small number of mammary epithelial cells retaining 3H-Tdr label, most were ER-positive luminal cells and only a few were ER-negative basal cells. This study indicates that pubertal growth of the mammary gland comprises division of ER-negative cap cells and of both ER-negative and ER-positive cells in the body of the TEBs and elongating mammary ducts. Similarly, estrogen-driven proliferation of ER-negative and ER-positive luminal cells and ER-negative basal cells maintains the differentiated mammary gland in the adult mouse.