The entire second thoracic mammary glands of 4-week-old BALB/c female mice primed with oestradiol plus progesterone were cultivated in organ culture medium containing the 'growth-promoting' hormone combinations: insulin, prolactin, growth hormone, oestradiol, progesterone and aldosterone or insulin, prolactin and aldosterone. Full lobulo-alveolar development was induced after 5–6 days of incubation and could be maintained for 15–16 days in organ culture in medium containing either hormone combination. After the initial 5–6 days in the 'growth-promoting' medium, subsequent cultivation of the glands in a medium with the 'lactogenic hormones', insulin, prolactin plus cortisol, led to accumulation of 'milk-like' secretory material in the ductal and alveolar lumina. Incubation of the lobulo-alveolar gland in medium with insulin alone for 7–9 days resulted in complete regression of the alveoli leaving only a ductal parenchyma. Incubation in insulin, prolactin, growth hormone or insulin plus the steroid hormones for 7–9 days led to considerable alveolar degeneration without a complete regression. The results indicate that both pituitary and steroid hormones are essential for development and maintenance of mammary alveoli; insulin can only sustain the basal ductal structure
