Heregulin (HRG), a ligand of ErbB receptor tyrosine kinases, is a potent mitogenic factor for breast cancer cells. Prolactin (PRL) has also been reported to regulate proliferation in breast cancer cells through its receptor, a member of the type I cytokine receptor family. Cytokine receptors are potent mitogens in hematopoietic cells, where they also override DNA damage-induced growth arrest checkpoints through activation of a phosphatidylinositol-3 kinase (PI3K) signaling pathway. In this study, we assessed the effect of gamma-irradiation on the mitogenic activity of HRG and PRL in breast cancer cells. HRG and PRL enhanced the proliferation of non-irradiated breast cancer cell lines in association with their ability to activate PI3K signaling pathways. Both growth factors also overrode irradiation-induced growth arrest in T47D cells, which resulted in decreased viability after irradiation. An inhibitor of PI3K, LY294002, abrogated growth factor-induced proliferation and the activity of cell cycle-dependent kinases in non-irradiated and irradiated cells. Thus, growth factors acting through distinct receptor families share a similar PI3K-dependent ability to promote proliferation and override DNA damage-induced growth arrest in breast cancer cells. These observations also suggest that selective activation of PI3K-dependent signaling can enhance radiosensitivity in breast cancer cells.