Expression of growth differentiation factor 9 (GDF-9), an apparent regulator of follicular development, reportedly differs between compartments of the rodent (oocytes) and human (oocytes and granulosa cells) ovary. To further characterize GDF-9 expression and action in the primate periovulatory follicle, adult female rhesus monkeys received recombinant human gonadotropins to promote multiple follicular development. Whole ovaries or follicular aspirates were obtained before and at various times after administration of an ovulatory dose of hCG; time points for tissue collection spanned the 40-h periovulatory interval. GDF-9 mRNA was detected by reverse transcription polymerase chain reaction assay in each oocyte and every granulosa cell sample examined, but granulosa cell GDF-9 mRNA levels did not change across the periovulatory interval. GDF-9 was also detected in follicular fluid using Western blotting; GDF-9 protein concentration in follicular fluid did not change across the periovulatory interval. Immunocytochemical staining for GDF-9 indicated that oocytes of both small and large antral follicles were positive for GDF-9. GDF-9 immunoreactivity was also present in cumulus granulosa cells and mural granulosa cells near the cumulus stalk. When granulosa cells from preovulatory follicles were exposed to recombinant GDF-9 in culture, GDF-9 increased vascular endothelial growth factor levels in culture medium. These data demonstrate that the cells of the primate periovulatory follicle both produce and respond to GDF-9. However, GDF-9 expression and action differ between rodent and primate follicles, suggesting a possible regulatory role for GDF-9 that is unique to the primate follicle.