Immature bovine oocytes were cultured to investigate whether the addition of FCS(10% or 20% ), CS (10%or 20% ) or BSA(5mg/ml) to culture medium with or without HEPES and co-culture with granulosa cells affect the frequency of in vitro maturation of extrafollicular bovine oocytes. After culture, the maturation rates were examined by the presence of 1st polar body and nuclear configuration. The maturation rate when FCS and CS as protein supplement were added to culture medium with or without HEPES was significantly higher than when BSA was added, and the maturation rate of extrafollicular bovine oocytes co-cultured with granulosa cells was higher than that cultured without granulosa cells, but there was no significant difference. FCS and CS were shown to be superior protein supplement when compared to BSA, and serum concentration, HEPES and co-culture with granulosa cells did not affect the in vitro-maturation of extrafollicular bovine oocytes.