Highly sensitive and specific radioimmunoassays to measure urinary FSH and LH were developed and applied to unextracted urine. The use of radioimmunoassays of gonadotrophins in 0.1 ml urine gave results which were significantly correlated (p < 0.001) with those obtained in acetone precipitates of urine. Gonadotrophin levels determined in unextracted urine were not influenced by variations in urine pH and by dilution. Urinary immunoreactive FSH appeared closely similar to pituitary standard, whereas LH was heterogeneous and partly different from the purified pituitary preparation. The urinary excretion of gonadotrophins was studied in 16 normal and 50 diabetic children and adolescents in relation to different time periods throughout day and night and to pubertal development. Diabetic subjects were shown to be similar to control subjects with respect to the daily excretion of gonadotrophins and creatinine. In both sexes, a sleep-wake pattern of FSH and LH excretion was observed, the lowest values being obtained during the night and the highest during the morning between 8 and 12 h. This circadian rhythm was found even in prepubertal children. Marked changes seem to occur throughout puberty: gonadotrophin levels excreted in morning urine increase mostly until midpuberty (stage 3) whereby the circadian pattern is most apparent; night excretion of gonadotrophins remains at a prepubertal level until midpuberty and slightly increases in late puberty (stage 4–5). Consequently, the differences between night and morning values of gonadotrophin excretion are less evident in late pubertal subjects (stage 5), especially in girls.