A single fraction of 50 Gy extracorporeal irradiation, as a modality of limb-sparing operation, has been used to achieve tumor necrosis in osteosarcoma. Although this modality of radiation therapy preserving the mobility of a joint is commonly practiced, the precise knowledge on the radiobiological response of osteosarcoma cell has remained to be elucidated. We therefore observed whether a single high dose irradiation caused apoptosis in osteosarcoma cells and whether the commitment to apoptosis was associated with cell kinetics. We also investigated radiation dose response along the time course for development of apoptosis following single high dose irradiation. The morphologic change in apoptosis was observed by fluorescence with Hoechst 33258 and the degree and the fraction of cells by flow cytometry. Irradiation of osteosarcoma cells with 10, 30 and 50 Gy resulted in chromatin condensation and apoptotic body formation. The degree of apoptosis in osteosarcoma cells was $29.5{pm}3.56%$, $39.9{pm}4.83%$ at 24 and 48 hours after 10 Gy irradiation ; $41.1{pm}3.93%$, $66.9{pm}5.21%$ at 24 and 48 hours after 30 Gy irradiation ; and $48.0{pm}3.69%$, $75.6{pm}4.65%$ at 24 and 48 hours after 50 Gy irradiation. The fraction of cells in cell-cycle kinetic was $39.2{pm}4.3%$ in G2/M, $22.1{pm}4.65%$ in G1 at 24 hours after 10 Gy irradiation ; $51.0{pm}4.3%$ in G2/M, $20.4{pm}4.7%$ in G1 at 48 hours after 10 Gy irradiation ; $40.3{pm}3.9%$ in G2/M, $26.1{pm}4.7%$ in G1 at 24 hours after 30 Gy irradiation ; $59.2{pm}3.9%$ in G2/M, $5.9{pm}5.1%$ in G1 at 48 hours after 30 Gy irradiation ; and $44.3{pm}4.2%$ in G2/M, $21.1{pm}3.5%$ in G1 at 24 hours after 50 Gy irradiation. The fraction of cells at 48 hours after 50 Gy irradiation could not be observed because of irradiation induced cell death of most of cells. All values for irradiated cells showed accumulation in G2/M phase and reduction in G1 phase, irrespective of irradiation dose. The results suggest that a single fraction of high dose irradiation with 50 Gy results in accumulation of cells at G2/M phase, leading to apoptosis.