BACKGROUND: In horses, the mechanisms behind ovarian follicle growth and oocyte maturation remain largely unknown. In other species, oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) have been related to the acquisition of developmental competence and to interaction with granulosa cells for the regulation of follicle development. This study assessed the expression and localization of GDF9 in the equine ovary, and its possible relationship with granulosa cell function. RESULTS: Using custom-made antibodies, GDF9 protein was localized in oocytes from the primary follicle stage onwards. Together with BMP15, its intrafollicular concentration was higher in small antral follicles compared to larger ones (P < 0.05). Negative correlations were observed between intrafollicular BMP15 concentration and estradiol sulfate (E2S) (r = -0.36, P = 0.048), as well as between BMP15 and E2S/P4 ratio (r = -0.37, P = 0.046). In vivo, equine granulosa cells showed increasing mRNA expression of genes involved in steroidogenesis (STAR and HSD3B2) and cell proliferation (KI67) with increasing follicle size, while expression of GDF9 and of apoptosis-related genes (BCL2 and CASP3) were not affected by follicle size. Simultaneous stimulation of granulosa cells in vitro with IGF1 and cortisol significantly increased HSD3B2 and CYP19A1 transcriptional levels, as well as E2 concentration in culture media, while IGF1-induced P4 secretion was suppressed in the presence of cortisol. Blocking the stimulatory effect of IGF1 on E2, E2S and P4 by H89 was associated with increased GDF9 mRNA levels and reduced STAR, PCNA, KI67 and BCL2 mRNA expression. Significant negative correlations of GDF9 with STAR and PCNA mRNA, respectively, were seen in vivo and in vitro. CONCLUSIONS: Together, our results show GDF9 localization and expression in the equine ovary and a temporal relationship with steroidogenesis and cell proliferation within the surrounding granulosa cells. Moreover, results of the in vitro study suggest a supporting role of cortisol during follicle maturation. Our study sheds light on possible mechanisms for the regulation of ovarian function in horses using GDF9.