In some mammals, like cattle, sheep, and humans, the pool of resting, primordial follicles is formed during fetal life and little is known about the regulation of the early stages of ovarian follicular development. Bovine primordial and primary follicles first appear around day 90 and 140 of gestation, respectively (gestation length = 280 days). Estradiol inhibits activation (i.e. initiation of growth) of newly formed bovine primordial follicles in vitro and secretion of estradiol by bovine fetal ovaries from around 83 to 140-day-old fetuses declines precipitously with age, suggesting an inhibitory role for estradiol in follicle activation in vivo. If inhibition of activation of newly formed bovine primordial follicles by estradiol in vitro mimics a normal developmental mechanism, we would expect the inhibition to be reversible. Therefore, the first objective of the present study was to test the hypothesis that the inhibitory effect of estradiol on follicle activation is reversible. Pieces of fetal ovarian cortex (4 pieces/treatment/fetus; n = 2 fetuses; day 100 and 120 of gestation) were cultured in serum-free medium supplemented with ITS+ (control medium) for 10 or 14 days (controls), with estradiol (10-6 M) for 10 days, or with estradiol for 10 days and then in control medium for 4 more days. On day 0 (time 0 controls) and after culture, cortical pieces were fixed and embedded in paraffin for serial sectioning and sections were stained with H&E for morphmetric analysis. On day 0, cortical pieces contained mostly primordial follicles (99.95%), whereas after culture the percentages of primary follicles in all cultures, except cultures treated with estradiol, were significantly higher than in day 0 controls (P < 0.05), suggesting that follicle activation occurred in vitro. There was a higher percentage of primary follicles in day 14 controls than in day 10 controls (75.4 ± 4% vs. 41.7 ± 8.3%; P < 0.05), suggesting that the number of follicles capable of activating increased with time in culture. The percentage of primary follicles in cortical pieces cultured with estradiol was lower compared with day 10 or 14 controls, (12 ± 4.6 vs. 41.7 ± 8.3 and 75.4 ± 4%, respectively; P < 0.05), consistent with our previous results showing that estradiol inhibits follicle activation. The inhibitory effects of estradiol on activation were reversed by culturing cortical pieces in control medium from day 10-14 (12 ± 4.6 vs. 66.9 ± 13.8% primary follicles; P < 0.05). Since we showed previously that the capacity of bovine primordial follicles to activate is associated with the achievement of meiotic arrest in their oocytes at the diplotene stage of prophase I, we next examined whether the inhibition of follicle activation by estradiol is associated with inhibition of the progression of meiotic prophase I. The results showed a higher percentage of primordial follicles with oocytes in pre-meitotic arrest (pre-diplotene) stages in cultures treated with estradiol than in day 10 or day 14 controls (92.5 ± 7 vs. 43.2 ± 5 or 13.5 ± 13, respectively; P < 0.05). In summary, the capacity of newly formed bovine primordial follicles to activate increases with time in culture. Inhibition of activation of newly formed bovine follicles by estradiol is reversible and is associated with inhibition of the progression of meiotic prophase I.