The recent availability of both cordocentesis, a low risk and effective technique for fetal blood sampling, and ultrasensitive/highly specific two-site immunofluorometric assays (IFMA) for pituitary and chorionic glycoprotein hormone (I-LH, I-FSH, and I-CG) measurement prompted us to study the maturation of hypothalamic-pituitary-gonadal function in 114 normal human fetuses (49 females and 65 males) from 17-40 weeks gestation. The subjects were selected from 216 consecutive cordocenteses carried out for rapid karyotyping and diagnosis of fetal infection or hematological disorders. In addition, FSH bioactivity (B-FSH) was measured by rat Sertoli cell aromatase induction assay, glycoprotein hormone alpha-subunit (alpha-SU) by RIA, and circulating free testosterone (fT) by direct analog technique. No significant cross-reactions were recorded in the different measurement methods. In particular, alpha-SU did not interfere in any IFMA, and CG cross-reactivity in LH IFMA was 0.5%. Circulating I-LH, I-FSH, and B-FSH levels at 17-24 weeks gestation were significantly higher in female than in male fetuses (I-LH, 48 +/- 4 vs. 6.3 +/- 0.7 U/L; I-FSH, 35 +/- 2 vs. 0.7 +/- 0.1 U/L; B-FSH, 131 +/- 17 vs. 43.4 +/- 5.4 U/L). During the last weeks of gestation, a significant decrease in I-LH and I-FSH levels was seen in both female and male fetuses (I-LH, 0.24 +/- 0.05 and 1.0 +/- 0.3 U/L; I-FSH, 0.45 +/- 0.1 and 0.5 +/- 0.1 U/L), while serum B-FSH remained elevated, but the previously recorded difference between sexes disappeared (54.3 +/- 7.2 and 58.7 +/- 7.3 U/L). Circulating I-CG and alpha-SU levels at midgestation were elevated in both female and male fetuses (I-CG, 117 +/- 29 and 191 +/- 44 U/L; alpha-SU, 143 +/- 16 and 105 +/- 9 micrograms/L, respectively) and decreased thereafter (I-CG, 42 +/- 9 and 26 +/- 6 U/L; alpha-SU, 60 +/- 15 and 37 +/- 6 micrograms/L). Serum fT levels at midgestation were significantly lower in females than in males (4.3 +/- 0.9 vs. 10.0 +/- 0.8 pmol/L) and increased until term, when the difference between sexes disappeared (16.2 +/- 1.8 vs. 17.6 +/- 1.6 pmol/L).(ABSTRACT TRUNCATED AT 400 WORDS)