Two experiments were performed to study chromosomal anomalies. In Experiment 1, chromosome complements of the inner cell mass (ICM) were investigated that had been separated immunosurgically from 169 and 83 bovine blastocysts cultured either in vitro or in vivo in rabbit oviducts from the four-cell stage following in vitro fertilization of in vitro-matured follicular oocytes. The incidence of embryos with chromosomal anomalies in the ICM cells was 18.2% (4/22) for in vitro cultured embryos and 22.2% (4/18) for in vivo cultured embryos and did not differ significantly from those of entire embryos. One haploid (4.5%), two triploid (9.1%) and one 2N/3N (4.5%) in vitro and three 2N/3N (16.7%) and one 2N/4N mosaic in vivo, respectively, were observed in the two culture systems. In Experiment 2, the origin of chromosomal anomalies observed in ICM cells was investigated using early bovine embryos derived from the same bull semen used in Experiment 1. Both the 2N/3N and 2N/4N anomalies were also observed in two-cell embryos. These results indicate that chromosomal anomalies were not restricted to ICM cells and that the 2N/3N anomaly in ICM cells may have been fertilization-derived chimera.