In-vitro maturation (IVM) of human ovarian follicles and oocytes could benefit infertile women, and allow the development of in-vitro systems for the study of human follicular development. Little is known about the initiation of growth of primordial follicles and the regulation of early folliculogenesis. An ovarian tissue-slice culture system was used to examine the effects of media composition, follicle stimulating hormone (FSH) and serum substitution on the development of small human follicles in vitro. Human ovarian cortex biopsies were cut into small pieces and cultured for 5, 10 or 15 days. Control (non-cultured) and cultured tissue was fixed, serially sectioned, and stained. The follicles contained within the tissue pieces were counted, measured, and assessed for stage of development and viability. Comparison of the ability of alpha-minimum essential medium (alpha-MEM), Waymouth's, or Earle's balanced salt solution (EBSS) culture media (all with 10% human serum) to support follicle growth demonstrated significantly increased initiation and growth of follicles in alpha-MEM during the first 10 days of culture. The supplementation of alpha-MEM with 300 mIU/ml FSH significantly reduced levels of atresia and increased the mean diameter of healthy follicles. Follicles in tissue cultured for 10 days with human serum albumin and ITS (insulin/transferrin/selenium mix) were significantly larger, more developed and showed significantly less atresia than those cultured with serum alone. Primordial to small preantral follicles can be grown under serum-substituted conditions in tissue-slice culture, and are responsive to FSH, which is thought to be acting mainly as a survival factor at these early stages.