Insulin-like growth factor-binding protein (IGFBP) production by the ovine ovary was examined using Western ligand blots and immunoblots. During follicle development, the follicular fluid content of the 39- to 42-kDa binding protein (IGFBP-3) increased, whereas that of the 34-kDa (IGFBP-2), 32-kDa, 30-kDa, and 25-kDa (IGFBP-4) binding proteins decreased. The granulosa and theca cell cultures produced different complements of binding proteins. IGFBP-2 and the 30- and 32-kDa binding proteins were produced by granulosa cells, and there was no obvious effect of the size of follicle from which the cells were isolated. Both FSH and insulin-like growth factor (IGF-I) were necessary for maximum production of IGFBP-2. The main binding proteins detected in theca cell cultures were IGFBP-4 and -2. The IGFBP-4 concentration increased when the cells were exposed to LH. In contrast, IGFBP-2 concentration in theca cell-conditioned medium decreased when the cells were incubated with LH. Theca cell cultures produced more IGFBP-4 when the cells were isolated from small follicles as opposed to large follicles. Neither granulosa nor theca cell cultures produced significant quantities of IGFBP-3, and the source of this binding protein in the follicular fluid from large ovine follicles is probably the circulation. The results indicate that IGFBP production in the developing ovine ovarian follicle is dependent on both cell type and follicle size and is regulated by IGF-I and gonadotropins.