Measurement of estradiol-17β, testosterone, and androstenedione in follicular fluid obtained from groups of antral follicles of known diameter and stage of morphological atresia indicates two phases of steroidogenesis during nonatretic growth. In pools of fluid obtained from follicles of 1 to 3 mm diameter, a transient fourfold increase in testosterone concentrations form 99 to 420 nM was observed soon after antrum formation, whereas concentrations of androstenedione and estradiol-17β ranged from 4 to 16 and 10 to 47 nM, respectively. In the pooled fluid of nonatretic follicles of greater than 3 mm diameter, concentrations of estradiol-17β exceeded that of either testosterone or androstenedione, and increased from 9 to 341 nM as follicular diameter increased. Testosterone concentrations progressively decreased from 247 to 56 nM, and the concentration of androstenedione decreased from 22 to 7 nM as follicular diameter increased beyond 3 mm. Concentrations of all three steroids in pools of fluid from early atretic follicles were not significantly different from those of nonatretic follicles, but in the later stages of atresia concentrations of testosterone remained elevated and those of estradiol stayed low at all stages of growth. Data obtained from individual follicles confirmed the initial finding that a reduction in the ratio of estrogen to total follicular androgen in pools of follicular fluid was associated with atresia of all sizes of follicles. We propose that the changes in patterns of steroidogenesis which accompany atresia reflect some of the earliest changes in the functional capacity of follicles undergoing atresia, but are not necessarily the primary cause of atresia.