In response to γ-irradiation (IR)-induced DNA damage, activation of cell cycle checkpoints results in cell cycle arrest, allowing time for DNA repair before cell cycle re-entry. Human cells contain G1 and G2 cell cycle checkpoints. While G1 checkpoint is defective in most cancer cells, commonly due to mutations and/or alterations in the key regulators of G1 checkpoint (for example, p53, cyclin D), G2 checkpoint is rarely impaired in cancer cells, which is important for cancer cell survival. G2 checkpoint activation involves activation of ataxia telangiectasia-mutated (ATM)/ATM- and rad3-related (ATR) signalings, which leads to the inhibition of Cdc2 kinase and subsequent G2/M cell cycle arrest. Previous studies from our laboratory show that G2 checkpoint activation following IR exposure of MCF-7 breast cancer cells is dependent on the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signaling. As HER receptor tyrosine kinases (RTKs), which have important roles in cell proliferation and survival, have been shown to activate ERK1/2 signaling in response to various stimuli, we investigated the role of HER RTKs in IR-induced G2/M checkpoint response in breast cancer cells. Results of the present studies indicate that IR exposure resulted in a striking increase in the phosphorylation of HER1, HER2, HER3 and HER4 in MCF-7 cells, indicative of activation of these proteins. Furthermore, specific inhibition of HER2 using an inhibitor, short hairpin RNA and dominant-negative mutant HER2 abolished IR-induced activation of ATM/ATR signaling, phosphorylation of Cdc2-Y15 and subsequent induction of G2/M arrest. Moreover, the inhibition of HER2 also abrogated IR-induced ERK1/2 phosphorylation. In contrast, inhibition of HER1 using specific inhibitors or decreasing expression of HER3 or HER4 using short hairpin RNAs did not block the induction of G2/M arrest following IR. These results suggest an important role of HER2 in the activation of G2/M checkpoint response following IR.