The present study examined whether the timing of the first cleavage has an effect on the in vitro and in vivo developmental potential of nuclear-transferred mouse oocytes receiving embryonic stem cells. First, the timing of the first cleavage and the developmental potential of nuclear-transferred oocytes were examined every hour from 12 to 24 h after the start of culture and compared with in vitro-fertilized oocytes. The developmental potential of in vitro-fertilized oocytes decreased gradually according to the time required for cleavage (84% (32/38) for 15 h to 50% (1/2) for 20 h), but intermediate-cleaved (15-16 h) nuclear-transferred oocytes had a higher potential to develop into blastocysts (55% (17/31) to 67% (45/67) versus 0-43% (6/14)]. Second the nuclear-transferred oocytes were divided into three groups according to the timing of the first cleavage; each group was cultured to blastocysts in vitro, and then transferred to recipients. The potential of intermediate-cleaved oocytes (15-16 h) to develop into blastocysts was significantly higher than fast-cleaved (before 15 h) and slow-cleaved (after 16 h) oocytes (65, 46, and 37%). The proportion of fetuses on Day 10.5 of pregnancy was highest in the intermediate-cleaved group (4 versus 2 and 1%, respectively) and a full-term fetus was obtained from this group. The present study demonstrated that the timing of the first cleavage could be used to determine the potential of nuclear-transferred oocytes with embryonic stem cells to develop to the blastocyst stage in vitro, but not to determine post-implantation viability after transfer to recipients.