AbstractRetinol (vitamin A) is essential for reproduction, and retinoids have been suggested to play a role in ovarian steroidogenesis, oocyte maturation, and early embryonic development. Retinol is transported systemically and intercellularly by retinol‐binding protein (RBP). Within the cell, cellular retinol‐binding protein (CRBP) functions in retinol accumulation and metabolism. Since the actions of retinoids are mediated, in part, by retinoid‐binding proteins, the objective of this study was to investigate cell‐specific expression of RBP and CRBP in the bovine ovary. Immunocytochemical analysis (ICC) localized RBP to the thecal and granulosa cell layers of antral and preantral follicles with the most intense staining in the cells of large, healthy follicles. The tunica adventitia of arterial blood vessels also exhibited RBP staining. Immunostaining of CRBP was most intense in the granulosa cells of preantral follicles and present, but diminished, in thecal and granulosa cells of antral follicles. Within the corpus luteum, both proteins were observed in large luteal cells, but only RBP was observed in small luteal cells. Northern blot analysis demonstrated that thecal and granulosa cells from antral follicles and luteal tissue expressed RBP and CRBP mRNA. Synthesis and secretion of RBP by thecal cells, granulosa cells, and luteal cells were demonstrated by immune‐complex precipitation of radiolabeled RBP from the medium of cultured cells or explants, followed by SDS–PAGE and fluorography. Follicular fluid was collected from small (<5 mm) and large (8–14 mm) follicles, pooled according to follicular size, and analyzed for retinol, RBP, estradiol‐17β, and progesterone. Concentrations of retinol, RBP, and estradiol were greater in the fluid of large follicles. Results demonstrate retinoid‐binding protein expression by bovine ovaries and provide physical evidence that supports the concept that retinoids play a role in ovarian function. Mol. Reprod. Dev. 64: 261–269, 2003. © 2003 Wiley‐Liss, Inc.