Breast cancer has a predilection for spreading to bone. The mechanism of preferential metastasis of breast cancer to bone is unknown. We hypothesize that breast cancer cells that develop bone metastases have the capacity to facilitate their colonization in bone. To examine this hypothesis, we established bone-seeking (MDA-231BO) and brain-seeking (MDA-231BR) clones of the human breast cancer cell line MDA-MB-231 by repeated sequential passages in nude mice and in vitro of metastatic cells obtained from bone and brain metastases, respectively. These clones were examined for distinguishing biological characteristics and compared with the MDA-231 parental cells (MDA-231P) in vivo and in vitro. Both the MDA-231BR and the MDA-231BO showed identical tumorigenicity to MDA-231P at the orthotopic site. MDA-231P that was inoculated into the heart developed metastases in bone, brain, ovary, and adrenal glands. On the other hand, MDA-231BO exclusively metastasized to bone with larger osteolytic lesions than MDA-231P. MDA-231BR exclusively disseminated to brain and failed to develop bone metastases. In culture, MDA-231BO produced greater amounts of parathyroid hormone-related protein (PTH-rP) than MDA-231BR and MDA-231P in the absence or presence of transforming growth factor β (TGF-β). Furthermore, the anchorage-independent growth of MDA- 231BO in soft agar was not inhibited by TGF-β, whereas TGF-β profoundly inhibited the growth of MDA-231P and MDA-231BR. Insulin-like growth factor I (IGF-I) markedly promoted the anchorage-independent growth of MDA-231BO, whereas marginal or no stimulation was observed in MDA-231BR or MDA-231P, respectively. Our data suggest that these phenotypic changes allow breast cancer cells to promote osteoclastic bone resorption, survive, and proliferate in bone, which consequently leads to the establishment of bone metastases.