Ionizing radiation is the exogenous agent best proven to induce breast cancer. c-erbB2/neu amplification and overexpression are known to occur in breast cancer and are correlated with aggressive tumor growth and poor prognosis. We have developed simian virus 40-immortalized cell lines from normal human breast epithelial cells (HBECs) with luminal and stem-cell characteristics. In this study, we examined whether x-rays and a mutated neu oncogene are capable of inducing tumorigenicity in these cells. The results indicated that x-rays were effective in converting immortal non-tumorigenic HBECs to weakly tumorigenic cells that then could be transformed to highly tumorigenic cells by the neu oncogene. The in vitro growth of these tumorigenic cells was significantly faster than that of the parental non-tumorigenic cells in growth factor- and hormone-supplemented or -depleted media. The neu oncogene, however, had no tumorigenic effect on immortal non-tumorigenic cells. The expression of p185(c-erb82/neu) was elevated in neu-transduced immortal or weakly tumorigenic cell lines. However, only in the latter was p185(c-erbB2/neu) found to be phosphorylated at tyrosine residues. Thus, x-rays appear to induce a genetic alteration that confers weak tumorigenicity on immortal HBECs and interacts with p185(c-erbB2/neu) directly or indirectly to give rise to fast-growing tumors.