We investigated whether or not the mitochondrial genotypes affect radiation-induced micronucleus (MN) formation. For that purpose, the rho+, KT1 and rho0 human osteosarcoma cell lines were used, which carry the wild-type mitochondrial DNA (mtDNA), the tRNALys mutant mtDNA and no mtDNA, respectively. Despite no significant difference in the clonogenic radiosensitivity, the rho+, KT1 and rho0 cells exhibited high, intermediate and low radiosensitivities, respectively, to the MN induction in cytokinesis-blocked binucleated cells. Such differential MN inductions were correlated with high, intermediate and low levels of cellular ATP in the rho+, KT1 and rho0 cells, respectively, but not exactly with ROS production. Antimycin A that inhibits the respiratory complex III reduced the rate of radiation-induced MN induction in the rho+ and KT1, but not rho0 cells. Thus, the functional status of the mtDNA to produce ATP appears to play a significant role for radiation-induced MN.