Ovarian growth and development are critically dependent upon the influence of endogenous estrogens, and both are highly regulated during the reproductive cycle. The observation that estrogen-receptor-α-deficient mice still exhibit follicular growth and development, together with other evidence, suggests that responsiveness of the ovary to estradiol occurs predominantly through the second estrogen receptor, ERβ. We characterized the physiological regulation of ERβ expression in ovarian follicles during the follicular phase of sheep that were synchronized for estrus during the breeding season with intravaginal progesterone implants (controlled internal drug release [CIDR] device; InterAg, Hamilton, New Zealand). Ovaries were removed at times corresponding to the early (EF) and late follicular phases (LF) of the ovine estrous cycle (12 h [n = 5] and 32 h [n = 5] after CIDR device removal, respectively). Sections of ovary were then hybridized with a cRNA probe corresponding to the 5′ region of ovine ERβ. ERβ mRNA expression within the granulosa layer of different size follicles (size classes: ≤3 mm, 3.1–4.0 mm, 4.1–5.0 mm, >5 mm) was quantified. ERβ mRNA expression varied both with follicle size (P < 0.01) and with cycle stage (P < 0.01). In EF ewes, the highest levels of ERβ mRNA expression were found in follicles ≤ 3 mm in size. ERβ mRNA expression declined progressively thereafter among the different size classes with lowest levels expressed in >5-mm follicles. By contrast, expression of ERβ mRNA in the 3.1- to 4.0-mm follicles of LF group was significantly higher than in the ≤3-mm size follicles and declined thereafter progressively to the >5-mm size levels as in the EF group. Furthermore, expression of ERβ mRNA in ≤3-mm size follicles of LF group was significantly lower than the corresponding size class in the EF group. Lower expression of ERβ mRNA in >5-mm follicle is suggestive of a down-regulation by the local estrogen milieu.