A model culture system for the study of follicular metabolism has been developed from existing methods of whole-follicle culture. The modified system mimics the in vivo growth and maturation of mouse ovarian follicles from primary to preovulatory stages and the modulating influences of the LH and FSH. This is the first study to demonstrate ovulation in vitro from individual ovarian follicles. The pattern of follicular lactate production relative to steroidogenesis was studied throughout in vitro follicular development over a period of 6 days. Twenty-four-hour samples of medium from individual follicles were analyzed for lactate and pyruvate by an automated analytical technique, and for estradiol and progesterone by an immunoenzymatic method. Follicles produced remarkably large quantities of lactate and estradiol during FSH-stimulated development in vitro. LH further stimulated lactate production but resulted in a significant decrease in estradiol secretion and an ovulation rate of 30%. Progesterone production was not detectable throughout the culture period, and follicles showed no evidence of pyruvate uptake. These findings demonstrate the validity of using this model culture system for the study of follicular metabolism and provide new information on the pattern of carbohydrate metabolism relative to steroidogenesis during follicular growth and maturation.