To investigate its inhibitory properties, bovine follicular fluid was collected at different stages of the estrous cycle from small, medium, and large follicles. Follicular fluids were incubated with germinal vesicle stage, bovine oocytes aspirated from small follicles. Nuclear maturation was determined at 24 h. Bovine follicular fluid inhibited the spontaneous maturation of bovine oocytes. The inhibition was reversed when follicular fluid was removed from oocyte cultures by 24 h. Follicular fluid varied in its ability to inhibit germinal vesicle breakdown according to follicle size and estrual stage. Follicular fluid from both small and medium follicles at estrus had the greatest ability to prevent germinal vesicle breakdown but became less potent postestrus. Follicular fluid from large follicles at estrus had less ability to inhibit germinal vesicle breakdown than fluid from small and medium follicles. Strong germinal vesicle breakdown inhibiting activity was present in large, probably atretic follicles at late metestrus, early diestrus, and mid diestrus. However, follicular fluid from large follicles had less germinal vesicle breakdown inhibiting activity at proestrus than follicular fluid from large follicles at late metestrus, early diestrus, and mid diestrus. This proestrual activity was similar to that in Graafian follicles at estrus.