Five continuous mouse mammary tumor cell lines and 12 clonal derivatives were established from tumors arising in BALB/c or C3H mice spontaneously or in response to viral (mammary tumor virus), hormonal (17.beta.-estradiol) or chemical [7,12-dimethylbenz(a)anthracene] stimuli in vivo. The cell lines were examined for the following in vitro growth parameters: plating efficiency, saturation density, population doubling times, colony formation on plastic surfaces and anchorage independent growth in methylcellulose. The majority of the mammary tumor cell lines were transplantable in syngeneic, immunocompetent mice and gave rise to tumors composed of epithelial cells. There was no growth parameter in vitro which invariably correlated with tumorigenicity in vivo. Most of the mammary tumor cell lines appeared to produce type C virus. Only 1, a C3H derivative, appeared to respond to stimulation by glucocorticoids with the enhanced production of type B virus. Analysis of the growth properties exhibited in culture by transformed mammary epithelial cells revealed marked differences from those previously reported for transformed fibroblasts.