Recent studies have suggested a bystander effect in nonirradiated cells adjacent to irradiated cells; however, the mechanism is poorly understood. In this study, we investigated the involvement of both extracellular nucleotides and activation of P2 receptors in cellular responses to γ radiation using human HaCaT keratinocytes. The concentration of ATP in culture medium was increased after γ irradiation (0.1–1.0 Gy), suggesting that radiation induces ATP release from cells. Intracellular Ca² concentration was elevated when conditioned medium from irradiated cells was transferred to nonirradiated cells, and this elevation was suppressed by apyrase (ecto-nucleotidase), indicating the involvement of extracellular nucleotides in this event. Further, we examined the activation of ERK1/2 by γ radiation and nucleotides (ATP and UTP). Both γ radiation and nucleotides induced activation of ERK1/2. Next, the effect of inhibitors of P2 receptors on radiation-induced activation of ERK1/2 was examined. The activation of ERK1/2 was blocked by suramin (P2Y inhibitor), MRS2578 (P2Y₆ antagonist) and apyrase. These results suggest that both released nucleotides and activation of P2Y receptors are involved in γ-radiation-induced activation of ERK1/2. We conclude that ionizing radiation induces release of nucleotides from cells, leading to activation of P2Y receptors, which in turn would result in a variety of biological effects.