Experiments were designed to determine optimal conditions for the cryopreservation of bovine embryos produced in vitro. In Expt 1, embryos were exposed for 1, 3 or 5 min to a vitrification solution consisting of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll and 10.26% (w/v) sucrose (EFS) and were subsequently vitrified. After warming in water at room temperature and diluting in a solution of 0.25 mol sucrose l-1, the in vitro survival rate in Ménézo-B2 medium was highest after exposure to EFS for 1 min. In Expt 2, embryos at day 7 and day 8 were vitrified after exposure to EFS for 1 min. The survival rate of embryos at day 7 was significantly improved, especially at the blastocyst and expanded blastocyst stage, when the Ménézo-B2 medium was supplemented with bovine oviduct epithelial cells (BOEC). Embryos at day 8 exhibited a significantly lower survival rate than did embryos at day 7 in both culture media. In Expt 3, one-step exposure of embryos to EFS for 1 min was compared with two-step exposure to 20% ethylene glycol for 3 min and EFS for 30-45 s. Embryos exhibited significantly higher survival and hatching rates after two-step vitrification, especially at the expanded blastocyst (89% and 69%, respectively) and the blastocyst stage (75% and 38%, respectively). In Expt 4, embryos were diluted in solutions of 0, 0.25 or 0.5 mol sucrose l-1 after two-step vitrification. There were no significant differences in the survival rates between the three dilution treatments.(ABSTRACT TRUNCATED AT 250 WORDS)