Conventional methods for isolation of cell lines from carcinomas suffer inherently from a lack of advantage for proliferation of transformed cells as opposed to contaminating fibroblasts and normal epithelial cells. To isolate cell lines from metastases of estrogen receptor-negative mammary carcinomas in dogs, we applied a novel method using medium supplemented with serum treated to inactivate growth factors. Under these conditions, autonomously growing tumor cells are selectively allowed to proliferate. In this way, four autonomously growing tumor cell lines were obtained from metastases of two dogs. Tumors formed from cells implanted in C3H nude mice closely resembled the original dog tumors, indicating that the main result of this selective procedure was suppression of normal cell proliferation. Serum treated to inactivate growth factors seems to be an important medium supplement for isolation of autonomously growing tumor cell lines, which may be valuable tools for future studies on regulation of cell proliferation in advanced hormone-independent mammary tumors.