In order to more clearly define the hormonal factors responsible for follicular growth, ovulation and steroidogenesis, a perifusion culture system was developed. This culture system was capable of exposing the ovaries to hormonal levels which mimic the 4-day estrous cycle of the rat. Using this culture system, ovaries were exposed to the in vivo levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol-17 beta and progesterone to within the mean +/- one standard deviation of the values reported by Butcher et al. (1974a). During the 4-day culture period, the diameter of the follicles gradually increased in a manner similar to that of cycling rats. After 4 days in vitro, the ovaries ovulated 4.3 +/- 0.6 oocytes/ovary as assessed by the presence of ruptured-partially luteinized follicles. This in vitro ovulation rate was not significantly different from the in vivo ovulation rate of 5.0 +/- 0.8 oocytes/ovary (P greater than 0.05). The pattern of follicular growth within cultured ovaries was similar to that of the cycling rat, although the number of small follicles recruited to grow was reduced. The steroid secretory patterns also were slightly different in the cultured ovaries. These data indicate that physiological ovarian responses which are similar to those of cycling rats can be induced within cultured ovaries. With further utilization of this culture technique, the precise role of gonadotropins, ovarian steroids and other hormonal agents can be examined in order to define specific ovarian functions which are controlled by each hormonal agent.