The present study focused on the level of Sr² activation required by mouse oocytes in round spermatid injection (ROSI) and analyzed the resultant embryos cytogenetically at the first cleavage and blastocyst stages. Mouse oocytes were divided into 3 groups: Group A, oocytes treated by activation using Sr² for 40 min before ROSI; Group B, oocytes treated by activation using Sr² for 1 h after ROSI; and Group C, oocytes treated by activation using Sr² for 5 h after ROSI. One round spermatid obtained from mature RFM/Ms-Rb(6.15) males was injected into each oocyte individually. The fertilization rate of oocytes in ROSI, the development rate of zygotes to the blastocyst stage and chromosomal normality in the resultant embryos were highest in Group A, suggesting activation using Sr² for 40 min before ROSI as the most appropriate treatment. At the first cleavage, many kinds of male-derived abnormalities, such as asynchrony and constitutive chromosome abnormalities etc., were observed in all groups. At the blastocyst stage, many parthenogenetic embryos, showing n, 2n and 2n/n with no translocated chromosome, were typically observed in all groups. Normal offspring were obtained by embryo transfer of the blastocysts derived from Group A, and their fertility after sexual maturity was confirmed by mating.