Using the irradiation system constructed for research in radiation biology, we have investigated the differences in the biological effects of in vitro irradiation by 65 MeV protons and by 137Cs gamma-rays. Survival curves were generated using V79 cells. The effects on biological parameters (SF2, RBE) of protons were greater than gamma-rays. Furthermore, 3H-thymidine incorporation in KOSC-3 cells, which display the p53 gene mutation, was inhibited by protons much more than by gamma-rays. On the other hand, in bleomycin-sensitive SCCKN cells, 3H-thymidine incorporation decreased more than in bleomycin-resistant SCCTF cells, however, both were inhibited by protons much more than by gamma-rays. In this study, the biological parameters and 3H-thymidine incorporation caused by 65 MeV protons were more severe than those caused by 137Cs gamma-rays.