Ionizing radiation (IR) induces chromosomal double-strand breaks (DSBs) repaired by two pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR), in mammalian cells. Cutting sections of DSBs induced by IR show structurally complex, not easy to connect by DNA ligase. Recently, we reported that repair of DSB introduced connectable I-SceI site, which is not real DSB induced by IR. Therefore, this connectable I-SceI repair model was not appropriate for real DSB repair. In this study, to clarify the relative contribution of each repair pathway for such complex DSBs, we developed a system to investigate the DSBs that occur in an endogenous single-copy human gene in human lymphoblastoid TK6 cells. Two DSBs, inversely arranged I-SceI restriction sites, were introduced at the either side of exon 5 of the TK gene (chromosome 17), and we named this cell line as TSCE206. We assessed for DSB repair, un-connectable I-SceI site repair, by transfection with the I-SceI expression vector in TSCE206. It was shown that the mutation frequency of TSCE206 expressed I-SceI was about 2-fold higher than TSCE105, which was introduced connectable I-SceI site. Molecular analysis showed the deletion size of TSCE206 mutants was larger than TSCE105 mutants. Furthermore, the mutants repaired by HR, which was rarely type in TSCE105, were observed in TSCE206. Thus, the repair pathway by NHEJ required making of large deletion to connect un-connectable DSB, which was radiation-like-double strand break. Additionally, we suggested that HR repair pathway might function in the situation, which was not easy to repair by NHEJ.