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Accurate measurements of 17beta-oestradiol and testosterone are important in clinical settings of pubertal disorders and hormone replacement therapy among children. Most assays for measurement of these steroids are optimised for fertility investigations in adults and are not sensitive enough for quantification in children. The primary aim of this thesis was to optimise the immunoassays available for measurement of these hormones in children and to develop reference ranges for healthy prepubertal and pubertal children. A second aim was to use the methods for improving induction of puberty with 17beta-oestradiol in girls.To increase the sensitivity of the original Spectria Estradiol Radioimmunoassay (RIA), the serum volume was increased and an extraction step using diethyl ether was added. These steps increased the sensitivity of the 17beta-oestradiol assay to 4.5 pmol/L. Twice the serum volume in the Spectria Testosterone RIA increased the sensitivity to 0.03 nmol/L.Using these methods for quantifying 17beta-estradiol and testosterone, reference ranges for healthy prepubertal and pubertal children have been established. Both girls (n=56) and boys (n=55) showed increasing levels of 17beta-oestradiol and testosterone throughout pubertal development. Girls demonstrated a diurnal secretion of the hormones prior to puberty. This diurnal secretion remains until one year after menarche. Boys demonstrated a similar diurnal secretion of serum testosterone before puberty and throughout puberty, but their diurnal rhythm of 17beta-oestradiol was not present until mid/late puberty. The diurnal rhythm of both hormones showed high levels in the morning and low levels in the evening. The patient group consisted of 15 girls with hypogonadism. A matrix patch containing natural 17beta-oestradiol was divided into parts for individual doses and attached on the girl s buttock nocturnally and serum 17beta-oestradiol was quantified. All girls in whom samples were taken during an 17beta-oestradiol dose of 0.08 - 0.12 µg/kg had serum concentrations that simulated the spontaneous levels as well as the diurnal pattern of serum 17beta-oestradiol in early pubertal girls. In conclusion, the changes in serum concentrations of 17beta-estradiol and testosterone, and the diurnal rhythm in healthy children have been assessed. With these new data it is possible to induce puberty in hypogonadal girls with physiological replacement therapy.