FSH plays an essential role in folliculogenesis and ovarian growth. However, cross-sectional studies have not shown an increase in bioactive FSH (B-FSH) during puberty. To eliminate intersubject variability, we used a longitudinal design and tested the hypothesis that B-FSH increases during puberty. Thirty normal, healthy girls were enrolled in a longitudinal study from pubertal stages I to IV. The subjects were evaluated at 6-mo intervals; each visit consisted of pubertal staging, bone age determination by x-ray, measurements of serum immuno-reactive FSH (I-FSH) and B-FSH (n = 14) or immunoreactive LH (I-LH) and bioactive LH (B-LH) (n = 18), and adrenal and ovarian steroids. All girls had clinical and hormonal characteristics of puberty. Both I-FSH and B-FSH levels were relatively elevated before puberty, whereas serum I-LH and B-LH were low. From pubertal stages I to III, there was a modest yet significant rise in serum I-FSH (p < 0.001) and serum B-FSH (p < 0.01). Serum I-LH and B-LH concentrations showed the expected increases with puberty (p < 0.001), with serum B-LH concentrations exhibiting a greater rise than I-LH (p < 0.001). Our results demonstrate that serum B-FSH and I-FSH increase during puberty. Relatively elevated B-FSH concentrations from early to midpuberty may be an important factor for ovarian growth while circulating LH and estrogen are still low. As puberty progresses, the continued and selective increase in LH induces a rise in estradiol and ultimately leads to ovulation.