Controversy exists about the validity of hyperfractionated radiation inverse of conventional fractionated radiation from clinical trials. It is necessary to design a real advantage of hyperfractionated radiation, compared with the conventional fractionation schedule of radiation treatment. To investigate the basic radiobiological response of hyperfractionated radiation, the 200µm HeLa S-3 spheroid were used in this study. The quantitative changes in growth rate, surviving fraction and cell kinetics of spheroids after various dose fraction schedules were compared. The data of growth rate and surviving fraction assays showed that repair and redistribution of cells during the radiation periods are important. Cell kinetics study showed a quite constant pattern of G,2 ,+ M block in the conventional radiaiton group, but a fluctuated pattern was seen in the hyperfractionated radiation groups. For further mimicking the clinical trail, a larger size of spheroid which have certain hyporic cells was necessary to being used in experiment.