The mechanisms controlling the initiation and early stages of follicular growth are poorly understood. Our laboratory developed a serum-free culture system that supports spontaneous and wholesale activation of primordial follicles in pieces of cortex dissected from the ovaries of fetal calves and fetal baboons. However, very few follicles activated in vitro progressed to the secondary stage. To determine whether androgens can promote the primary to secondary follicle transition, pieces of fetal bovine ovarian cortex were cultured in serum-free medium in the absence or presence of testosterone (T, 10⁻⁷ and 10⁻⁶ M) or estradiol (E₂, 10⁻⁶ M) for 10 days. Cortical pieces were then fixed and embedded in plastic for serial sectioning and morphometric analysis; fresh cortical pieces fixed on Day 0 served as uncultured controls. Freshly isolated cortical pieces contained mostly primordial follicles, whereas after 10 days in vitro, most primordial follicles had activated, differentiating into primary follicles as expected. Neither T nor E₂ affected the number of primordial and primary follicles compared with controls (P > 0.05). However, T (10⁻⁷ and 10⁻⁶ M) increased the number of secondary follicles (P < 0.05), whereas E₂ had no effect, suggesting that the effect of T was not due to conversion of T to E₂. In the second experiment, the optimal concentration of T for preantral follicle growth was determined. A range of lower doses of T (10⁻¹⁰–10⁻⁷ M) increased the number of secondary follicles in cultured cortical pieces in a dose-dependent manner, with 10⁻⁷ M T being the most effective (P < 0.05). In the third experiment, addition of a specific androgen receptor blocker, flutamide, inhibited the stimulatory effects of T on the primary to secondary follicle transition (P < 0.05), suggesting a receptor-mediated action of T. Localization of androgen receptors by immunohistochemistry revealed immunostaining for the androgen receptor in ovarian stromal cells and increasing immunoreactivity in follicle cells as follicular development progressed from primordial and primary to secondary to antral follicles, suggesting the involvement of the androgen receptor in bovine folliculogenesis. In summary, our results show that T promotes the growth of bovine follicles activated in vitro and suggest that its stimulatory effect is mediated through androgen receptors in the stroma and/or follicular cells.