Human breast epithelial cells dissociated from reduction mammoplasty specimens were embedded in two commonly used extracellular matrices, type I collagen gel or Matrigel, and subsequently transplanted subcutaneously into athymic nude mice. Histological sections from both types of recovered gels showed epithelial structures arranged as short tubules with some branching as well as preservation of epithelial cell polarity. Proliferation was studied in vivo by 5-bromo-2′-deoxy-uridine labeling followed by immunostaining of sections from recovered gels. Human breast epithelia embedded in collagen gel or Matrigel had similar proliferative activity. Cholera toxin, 17β-estradiol, and epidermal growth factor, when tested singly, were growth promoting, and in combination 17,β-estradiol and cholera toxin had an additive effect but 17,β-estradiol and epidermal growth factor were not additive. Our model system provides a means to study the endocrine control of normal human breast development.