A series of mouse mammary epithelial cell lines has been established by a protocol that gives highly reproducible results. The mammary epithelial cell lines, designated as FSK lines, were judged to be epithelial based on positive immunostaining for keratin-intermediate filaments, negative immunostaining for vimentin-intermediate filaments, hormonal induction of casein, and the ability to exhibit ductal and alveolar mammary morphogenesis in vivo. The FSK cell lines are dependent on epidermal growth factor and insulin in a low serum (1%) medium. Conditioned medium from spindle cell cultures replaced the requirement for serum and increased the growth of FSK3 and FSK4 4-5 times in collagen gels and 12-14 times in monolayer culture, respectively. Following injection into the mammary fat pad at passages 2-11, the FSK cell lines generated stable transplantable hyperplastic alveolar outgrowth lines. The in vivo outgrowth lines were judged as preneoplastic based on their stable alveolar morphology in vivo and an increased susceptibility for tumorigenesis. The FSK cell lines and their derivative in vivo outgrowth lines provide a new and potentially productive system to examine critical molecular alterations involved in the development of mammary preneoplasias. Furthermore, the reproducibility of the in vitro culture system provides the assurance that stable cell lines of mouse mammary epithelial cells can be generated easily and at will.