Our laboratory has developed twelve human breast cancer cell lines from primary and metastatic sites. In this report we demonstrate that eight of eight breast cancer cell lines examined exhibit constitutively tyrosine phosphorylated and enzymatically active endogenous pp125fak when grown in monolayer. The activation status of pp125fak in breast cancer cells in monolayer is significantly elevated over that exhibited by normal mammary epithelial cells cultured under the same conditions. Constitutive activation of pp125fak is the only characteristic so far studied that all of these breast cancer cell lines have in common. In contrast to HBC cells, tyrosine phosphorylation of pp125fak in HME cells was low or absent in monolayer culture but was induced to high levels by culturing the cells in Matrigel. Thus tyrosine phosphorylation and activation of pp125fak is a regulated process in normal mammary epithelial cells, but is constitutive in breast cancer cells. Finally, analysis of the ability of normal human mammary epithelial cells and breast cancer cell lines to grow under anchorage-independent conditions indicated that normal human mammary epithelial cells rapidly and uniformly lost viability when not substrate-attached, whereas all of the breast cancer cell lines survived for a 3-week culture period. Furthermore, a subset of the breast cancer cell lines grew to form large colonies under anchorage-independent conditions. Interestingly, pp125fak activation decreased dramatically in HBC cells cultured for two weeks in suspension, suggesting that activation of this kinase is not necessary for long-term growth under anchorage-independent conditions. These results suggest that constitutive activation of pp125fak results in preferential survival of human breast cancer cells under anchorage-independent conditions but that activation of pp125fak is not the sole mediator of anchorage-independent colony formation.