Bovine zygotes produced by in vitro maturation and fertilization (IVM-IVF) were selected according to the time of the first cleavage, and their subsequent developmental ability to the hatched blastocyst-stage and sex were assessed using conventional in vitro culture (IVC) conditions and PCR sexing. Of 669 presumptive zygotes cultured, 124, 227, 88 and 65 cleaving embryos were selected at 22, 26, 30 and 44 h post insemination (hpi), respectively. These embryos were transferred into separate drops of the same medium with cumulus cells and co-cultured for a total of 10 days. Significant correlation was observed between hatching rates and the time of the first cleavage (P<0.05): 56.5, 40.1, 21.6 and 4.6% of embryos cleaved at 22, 26, 30 and 44 hpi were developed to the hatched blastocyst-stage, respectively. Of 183 hatched blastocysts subjected to PCR for sexing, the sex of 171 embryos was successfully determined and the overall sex ratio was 51.5% (88/171). The sex ratios of early cleaving embryos (22 and 26 hpi) and late cleaving embryos (30 and 44 hpi) were 49.7% (74/149) and 63.6% (14/22), respectively. These ratios were not significantly different from the expected ratio of 1:1 and did not related to the time of the first cleavage. These data suggest that the interval from insemination to the first cleavage of embryos generated in vitro strongly affects their subsequent developmental potential in vitro, but not the sex difference.