Although many aspects of ovarian differentiation have been established, comparatively little is known about prenatal follicle formation and differentiation of bovine ovaries. The objective of this investigation was to study the role of the surface epithelium during the development of germ cell nests, germ cell cords and follicle formation in the fetal bovine ovary. Associated important proliferation and apoptotic features were further investigated. Additionally, the expression pattern of the S100 protein was detected. A strong increase of mitotic figures was detected in the surface epithelium, germ cell nests and germ cell cords of ovaries with a crown-rump length (CRL) of 13.0-58.0 cm. Oocytes were positively stained with S100 in bovine ovaries from fetuses with a CRL of 21.0 cm. The staining intensity enhanced parallel to increasing oocyte and follicle sizes during the ovary development. In later stages, a strong staining for S100 was observed in healthy oocytes in contradistinction to atretic oocytes where no expression of the S100 protein could be found. In conclusion, increasing mitosis index of surface epithelium cells, as well as oogonia directly beneath the surface epithelium, in combination with open surface connection during stages from a CRL of 11.0-94.0 cm of bovine fetal ovaries could play an important role in the period of time of ongoing folliculogenesis and derivation of granulosa cells. Additionally, S100-positive oocytes in primordial and later follicle stages joined by a high rate of Ki67-positive index in surrounding granulosa cells indicate that in the oocytes the S100 protein can perhaps be a useful marker for intact oocytes in bovine ovaries. Copyright (C) 2013 S. Karger AG, Basel