Abstract. The required concentration and time of vinblastine sulfate (a mitotic inhibitor) treatment were determined for successful cytogenetic diagnoses, such as normality and sex in bovine blastocysts, derived from in vitro fertilization. Chromosome preparations were made from blastocysts treated for 17 h, in media containing vinblastine sulfate, with various concentrations of 0, 30, 100, 200 and 300 ng/ml. Vinblastine sulfate was effective at a concentration of 100-300 ng/ml, with respect to the average numbers of metaphase plates and the mitotic indexes (% of mitoses) in the embryos. To determine the sufficient treatment time, embryos were placed in a medium containing 100 ng/ml vinblastine sulfate for 10, 17 and 24 h. The required time of vinblastine sulfate treatment was 10 h. The average length of the no.1 chromosome shortened with the prolongation of treatment time but not with significant difference. Most of the embryos (approximately 98%) treated with vinblastine sulfate had metaphase plates, and approximately 75% of them were numerically analyzable. A high incidence of polyploidy and a sex ratio showing male advantage were found in the present study.