The purpose of this study was to examine the effects of different fractions of bovine follicular fluid (bFF) and fetal bovine serum (FBS) as maturation medium supplements on development to the blastocyst stage of bovine oocytes matured and fertilized in vitro. Three bFF fractions obtained by ultracentrifugation were designated as the 1st, 2nd and 3rd fractions, and four FBS fractions, also obtained by ultracentrifugation, were designated as the 1st, 2nd, 3rd and 4th fractions. Cumulus-oocyte complexes (COCs) were divided into two groups: those with homogeneous ooplasm and those with heterogeneous ooplasm. COCs were cultured in basic maturation medium with a bFF fraction or FBS fraction. In oocytes with homogeneous ooplasm, the rates of development to the blastocyst stage with bFF-1st and bFF-2nd were significantly higher than that with bFF. In oocytes with heterogeneous ooplasm, the rate of development to the blastocyst stage with bFF-2nd was significantly higher than that with bFF. In oocytes with homogeneous ooplasm, the rate of development to the blastocyst stage with FBS-1st was significantly lower than that with FBS. In oocytes with heterogeneous ooplasm, the rate of development to the blastocyst stage with FBS-3rd was significantly higher than that with FBS. The results indicate that bFF-2nd and FBS-3rd, obtained by ultracentrifugation, are effective maturation medium supplements, as they promoted the development of high-quality matured bovine oocytes.