In-vitro growth of frozen-thawed human follicles is perceived as a potential option for restoring women's fertility. The aims of this study were: (i) to test the usefulness of a defined serum-free medium for growth of frozen-thawed human follicles; and (ii) to evaluate the expression of growth differentiation factor-9 (GDF-9) and anti-Müllerian hormone (AMH) in cultured follicles. Frozen-thawed ovarian cortical pieces from 7-, 12-, 25- and 27-year-old women were cultured for 0, 7, 14, 21 and 28 days. Follicle developmental quality was evaluated and expression of proliferating cell nuclear antigen (PCNA) (day 21), GDF-9 (days 14 and 28) and AMH (day 21) was assessed by immunohistochemistry. Primary follicles and enclosed oocytes underwent significant growth at the end of culture (P < 0.05). Cultured follicles from all patients studied reached the early secondary stage and a few follicles from two patients developed up to the secondary stage. The rate of atresia was variable throughout the culture periods. PCNA was expressed in the granulosa cells at all the different follicular stages. AMH and GDF-9 immunostaining were found respectively in the granulosa cells and oocytes after several weeks of culture. The transition from resting to growing follicles leading to the development of secondary follicles showed the normal expression patterns of GDF-9 and AMH.