The present study was designed to examine the ability of bovine oocytes, after germinal vesicle breakdown (GVBD), to be activated by sperm penetration and the sequence of sperm nuclear transformation. Bovine oocytes cultured for 8 h in maturation medium (tissue culture medium TCM-199 containing 10% fetal calf serum) were inseminated in Brackett and Oliphant's medium supplemented with bovine serum albumin (10 mg/ml), caffeine (5 mM) and heparin (10 micrograms/ml). When oocytes were transferred to the maturation medium 8 h after insemination and additionally cultured for 5-40 h at 39 degrees C in 5% CO2 in air, 71-76% of oocytes were penetrated and polyspermy (67-75%) was common. The proportions of penetrated oocytes that were activated significantly increased with the lapse of the additional culture time, reaching 88% and 87% by 25 and 40 h after additional culture, respectively. When compared with unpenetrated oocytes, significantly higher proportions of penetrated oocytes reached metaphase II or beyond 15 and 25 h after additional culture. After penetration, sperm nuclei were transformed into metaphase chromosomes and then to telophase chromosomes before the formation of male pronuclei. These results provide evidence that bovine oocytes acquire the ability to respond to sperm-mediated activation soon after GVBD.