Vitrification of bovine oocytes performed using the traditional, in straw system has not given satisfactory results. Although an alternative approach based on minimizing the volume of the vitrified sample has recently resulted in a much more promising survival rate of vitrified oocytes, we attempted to examine some additional factors influencing the survival and subsequent fertilization and development rates of bovine oocytes subjected to vitrification according to the minimum drop size approach. In total, 748 bovine, in vitro matured oocytes were vitrified using VS14 vitrification solution, containing 5.5-M ethylene glycol and 1.0-M sucrose after different pre-equilibration and equilibration protocols performed at 35 degrees to 37 degrees C. Experiment 1 showed no significant toxic effect during pre-equilibration treatments of oocytes in 2%, 4% or 6% ethylene glycol solutions, except the lower cleavage rate of oocytes exposed to 6% ethylene glycol (77.2% vs. 93.9% in control, P< 0.05). In Experiment 2, 12 to 15 min of pre-equilibration treatments in 0%, 1% or 2% ethylene glycol solutions were tested, followed by 30 or 45 sec of equilibration in VS 14 solution and vitrification in droplets of medium dropped directly into liquid nitrogen. The development rate of vitrified oocytes to the blastocyst stage tended to be higher after 30-sec equilibration treatment (9.5%, 13.9% and 13.8% in groups of oocytes pre-equilibrated in 0%, 1% or 2% ethylene glycol solutions, respectively). Experiment 3 tested pre-equilibration treatments in 0%, 1%, 2%, 3%, 4%, 5% or 6% ethylene glycol solutions, followed by 30-sec equilibration and vitrification in droplets. The highest cleavage, blastocyst and hatched blastocyst rates, which were not significantly different from control, were achieved in a group of oocytes pre-equilibrated in 3% ethylene glycol solution (76%, 30% and 15% vs. 89%, 42% and 21% in control, respectively). A healthy calf was born on Feb 22 1999, after transfer of 4 morula/blastocyst stage embryos developed from oocytes vitrified in droplets after pre-equilibration in 3% ethylene glycol solution. We conclude that gentle pre-equilibration of bovine oocytes in diluted, 3% ethylene glycol solution is an important factor improving the effectiveness of vitrification in droplets of bovine oocytes.