Development of the functional secretory epithelium in the mammary gland of the female mouse requires the elongation of the anlage through the mammary fat pad to form the primary/secondary ductal network from which tertiary ductal side-branches and lobuloalveoli develop. In this study we examined the hormonal requirements for the spatial development of the primary/secondary epithelial network and tertiary side-branches by quantifying ductal growth and epithelial cell proliferation in normal and hormone-treated BALB/c mice between 21 and 39 days of age. In normal mice, an allometric increase in ductal length commenced at 31 days of age and resulted in completion of the primary/secondary ductal network by 39 days of age. Concurrent with this allometric growth was a significant increase in cellular proliferation in the terminal end-buds (TEBs) of the ductal epithelium from 29 days of age, as determined by 5-bromo-2'-deoxyuridine (BrdU) incorporation. A level of cellular proliferation similar to that in the TEBs of 33-day-old control mice could be induced in the TEBs of 25-day-old mice following treatment for 1 day with estrogen (E), or progesterone (P) or both (E/P), indicating that both E and P were mitogenic for epithelial cells of the peripubertal TEBs. However, the period of allometric ductal growth in untreated mice did not correspond to an increase in serum E or P (which might have been expected during the estrous cycle). In addition, epithelial growth was not observed in mammary glands from 24-day-old mice that were cultured in vitro with E, P or E/P. In contrast to treatment with E, treatment with P promoted a dramatic increase, relative to control mice, in the number of tertiary branch points upon the primary/secondary ductal network. BrdU labeling of mammary glands from 24- 33-day-old mice pelleted with cholesterol (C), E, P or E/P confirmed the greater mitogenicity of P on the epithelial cells of the secondary/tertiary ducts as compared with C or E. Concurrent with these changes, localized progesterone receptor (PR) expression in clusters of cells in the ductal epithelium was associated with structures that histologically resembled early branch points from ductules. In conclusion, our results suggest that additional endocrine growth factor(s) other than E and P contribute to the development of the primary/secondary ductal network, and that P is responsible for the formation of tertiary side-branches in the mammary glands of mice during puberty.