The chronology of changes in levels of some proteins known to be altered during atresia of ovarian follicles was studied in ewes hypophysectomized at the end of the follicular phase of the estrous cycle. This study was performed by quantitative immunohistochemistry and zymography on large antral follicles (diameter > 3.5 mm) normally destined to ovulate, recovered 24, 36, or 72 h after pituitary ablation. The process of atresia was followed by comparing healthy follicles from intact ewes, with early atretic follicles recovered 24 h after hypophysectomy, clearly atretic follicles recovered 36 h after hypophysectomy, and late atretic follicles recovered 72 h after hypophysectomy. The earliest events of atresia induced by hypophysectomy were detected in theca and consisted of a strong decrease in levels of fibronectin (p < 0.001) and insulin-like growth factor II/mannose 6 phosphate (IGF-II/M6P) receptors (p < 0.05), occurring within the first 24 h following pituitary ablation. In intact animals, similar changes were observed in theca of early atretic follicles, suggesting that these changes may be important events involved in the onset of follicular atresia. In parallel, intrafollicular levels of matrix metalloproteinase-2 and -9 were shown to increase as early as 24 h after hypophysectomy, and a further increase was observed until 72 h after hypophysectomy. These early events were followed by the disappearance of P450 aromatase in granulosa cells 36 h after hypophysectomy (p < 0.05), and a progressive decrease in levels of P450 17alpha-hydroxylase in the theca interna and of gap junction protein connexin-43 in granulosa cells; these markers were still detectable in late atretic follicles 72 h after hypophysectomy. The increase in levels of fibronectin, type IV collagen, laminin, and IGF-II/M6P receptors within granulosa cell layers (p < 0.05) was significant only in late atretic stages, suggesting that these changes may be consequences rather than causes of atresia.