To evaluate the relative changes in serum bioactive (B) and immunoreactive (I) plasma gonadotropin concentrations during pubertal maturation, 28 healthy boys were enrolled at Tanner stage I and followed at 6-month intervals until achievement of Tanner stage V of pubertal maturation. At each visit, a careful interview, complete physical examination, sexual maturation staging, and bone age x-ray study were done, and a blood sample was obtained. Serum concentrations of PRL, dehydroepiandrosterone, and its sulfate, delta 4-androstenedione, estrone, estradiol, and testosterone (T) were determined by RIA. Samples from 20 boys were assayed for I-LH by RIA and for B-LH by the rat interstitial cell testosterone production assay, using 2 standards [Second International Reference Preparation-Human Menopausal Gonadotropin (2nd IRP-hMG) and LER 960]. Samples from 11 boys (3 from LH group and 8 others) were assayed for I-FSH by RIA and B-FSH by the rat Sertoli cell aromatase induction assay. The results were analyzed by regression analysis for B and I LH and FSH by Tanner stages of puberty, and by correlation of B to I LH and FSH as well as B and I LH and FSH to T. The results from both LH standards correlated well to each other (r = 0.967 and 0.882 for B- and I-LH, respectively), and the data are presented for 2nd IRP-hMG standard. In both groups of boys serum T concentrations increased progressively with pubertal development (P < 0.001). The boys bone age, testicular volume, serum T, dehydroepiandrosterone sulfate, dehydroepiandrosterone, delta 4-androstenedione, estrone concentrations correlated well with pubertal maturation, similar to previously published data and indicate that this group of boys had progressed through puberty in the expected normal manner. Mean serum I-LH concentrations increased progressively from Tanner stage I to V of puberty (P < 0.001), and serum B-LH exceeded the increase in serum I-LH levels. Mean serum I-LH concentrations were 2.0 +/- 0.1, 2.9 +/- 0.2, 4.7 +/- 0.4, 6.7 +/- 0.7, and 10.4 +/- 2.0 IU/L 2nd IRP-hMG whereas mean serum B-LH concentrations were 0.8 +/- 0.1, 2.2 +/- 0.2, 5.9 +/- 0.2, 10.3 +/- 1.2, and 22.3 +/- 3.8 IU/L 2nd IRP-hMG for Tanner stages I-V of puberty, respectively. This resulted in a progressive increase of LH B/I ratio with advancing pubertal maturation (P < 0.001).(ABSTRACT TRUNCATED AT 400 WORDS)