Recently the authors established a method for culturing mouse splenic T-lymphocytes with T-cell growth factor (TCGF) and feeder cells in vitro. Using this method, T-lymphocytes grow for approximately 14 days with population doubling times of 27-29 hr; cloning efficiencies (CEs) of mouse spleen cells ranged from three to twelve percent. Using this colony forming assay, in vivo and in vitro radiosensitivity of mouse splenic T-lymphocytes in the G0 phase and in vitro radiosensitivity of proliferating T-lymphocytes (cycling T-lymphocytes) were examined. For in vitro irradiation, the dose-survival curve of T-lymphocytes in G0 phase gave a D0 value of 0.99 Gy and a Dq value of 0.87 Gy and that of cycling T-lymphocytes gave a D0 value of 1.04 Gy and a Dq value of 0.19 Gy. For in vivo irradiation, the dose-survival curve of T-lymphocytes gave a D0 value of 1.01 Gy and a Dq value of 0.73 Gy. These results suggest that the recovering activity from sublethal damage of G0 T-lymphocytes was more effective than that of cycling T-lymphocytes. Furthermore, this colony forming assay system appears to be very useful for screening the effects of in vivo exposure to toxic and/or mutagenic agents and for comparing the effects of in vivo exposure with those of in vitro exposure to toxic agents as well as radiation.