To analyze a possible gonadal effect on the control of gonadotropin secretion during the prepubertal period, we have measured the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) serum concentrations in children with primary gonadal failure (PGF). We measured them using an ultrasensitive immunofluorometric assay (IFMA) in a single daytime serum sample and compared the results with those obtained with a radioimmunoassay (RIA) technique. The patients were 22 children with PGF (13 girls and 9 boys) aged 0.56-15.4 years and 58 normal children (28 girls and 30 boys) aged 0.08-16 years. In the normal group there were significant changes in serum LH and FSH concentrations in relation to sex and pubertal development. These changes were more evident especially in LH concentrations when using IFMA. We observed that during the prepubertal period the normal LH levels (mean ± SD) were detectable with this method at concentrations well below the limit RIA could detect (girls 0.026 ± 0.012 IU/1, and boys 0.025 ± 0.01 IU/1), while at the onset of puberty these LH levels rose significantly in both sexes (girls 1.0 ± 0.79 IU/1, boys 1.7 ± 0.7 IU/1; p < 0.01 vs. prepubertal group), reaching similar values to those observed in FSH concentrations (prepubertal girls 1.9 ± 0.89 IU/1, boys 0.73 ± 0.41 IU/1; early pubertal girls 3.1 ± 0.9 IU/1, boys 2.6 ± 1.3 IU/1). At prepubertal age, most PGF patients showed normal gonadotropin serum levels (particularly LH) when measured by RIA. However, these same samples – when measured by IFMA – showed LH and FSH levels clearly higher than normal in almost all – 10 of 12 – patients (PGF girls, n = 8, LH1.1 ± 1.0IU/1, FSH34 ± 30 IU/1; PGF boys, n = 4, LH0.13 ± 0.12IU/1, FSH 6.5 ± 5.7 IU/1). We conclude that, during the so-called ‘juvenile pause’ when the gonadotropin concentrations could be reliably measured, supranormal gonadotropin levels could be observed during a single daytime serum sample in patients with PGF. These findings suggest that during this period of life the gonads contribute to the negative feedback regulation of gonadotropin levels.