Bystander effects on unirradiated V79 cells were observed by irradiated conditioned medium (ICM) method and co−cultured with carbon−ion−irradiated V79 cells. The results showed that the colony formation efficiency of unirradiated cells is obviously decreased by ICM. After co−culture with carbon−ion−irradiated cells for some time, the colony formation efficiency of co−cultured cells was lower than expected results assuming no bystander effects. The micronucleus frequency and hprt gene mutation rate was almost the same as expected results. Cytotoxic factor(s), which was effective for cell growth but not for micronucleus and mutation on unirradiated cells, might be released by irradiated cells.