To assess the functional changes of theca and granulosa cells in antral follicles developing throughout pregnancy, individual follicles were isolated and incubated individually in vitro to measure the aromatization of testosterone or gonadotropin stimulation of estradiol and cAMP accumulation. Other follicles were processed to measure gonadotropin receptor content. Between Days 3-6, FSH and LH receptor content increased in granulosa cells, LH receptor content increased in theca cells, a progressive increase in gonadotropin stimulation of follicular cAMP was observed and the ability of follicles to aromatize testosterone increased. Nevertheless, the amount of estradiol accumulated by follicles incubated in the absence of exogenous testerone remained unchanged at 50 pg/follicle/4 h. Between Days 8-19, FSH and LH receptor content in granulosa cells and LH receptor content in theca cells decreased, estradiol accumulation by follicles incubated without testosterone declined to 10 pg/follicle/4 h and gonadotropin stimulation of cAMP was reduced. Nevertheless, the ability of follicles to aromatize testosterone was maintained at values (500 pg estradiol/follicle/4 h) seen on previous days. The preovulatory follicles developing between Days 19-23 were characterized by increased LH receptor in granulosa and theca cells, increased FSH receptor in granulosa cells and most notably by a marked increase in estradiol accumulation. On Day 23, 3500 ± 200 pg estradiol/follicle/4 h were accumulated either in the absence or presence of testosterone and between 10,000-14,000 pg estradiol/follicle/4 h were accumulated in the presence of oLH or hFSH. Since administration of LH-antiserum in vivo between Days 20-22 abolished estradiol accumulation by follicles incubated in the absence but not in the presence of testosterone, endogenous LH seems to exert a greater effect in stimulating androgen production than in maintaining aromatase activity. Gonadotropin stimulation of follicular cAMP production was also dramatically increased on Day 23 and appeared to be related to changes in granulosa cell function in addition to the increased density of gonadotropin receptors. Thus, although many small antral follicles possessing abundant aromatase activity are present continuously throughout pregnancy and although follicles between Days 3-6 exhibit some characteristics of 'preovulatory' follicles, the growth of the Graafian follicle appears to depend on increased follicular production of androgen, its immediate aromatization and the subsequent functional differentiation of granulosa cells.