A sensitive and reliable radioimmunoassay combined with celite column chromatography for specific determination of plasma testosterone (T) is described. The percentage binding was measured by equilibrium dialysis.In 28 normal newborns, total T concentration is significantly higher in male (68.1 ± 59.5) than in female newborn infants (12.0 ± 6.2) while binding of T increases rapidly after birth. Although there is no sex difference in TeBg capacity, levels of unbound T in the first two weeks of life are still significantly higher in male than in female infants.There was no sex difference in 62 normal prepubertal children studied, the total T concentration being 6.62 ± 2.46 and 6.58 ± 2.48 ng/100 ml in boys and girls, respectively; the binding of T and unbound T levels were identical in both.In 17 patients with Turner syndrome, total T concentration (13.6 ± 3.1 ng/100 ml) was significantly higher than in prepubertal children and increased (21.5 ± 2 ng/100 ml) under moderate estrogen therapy while the percentage of T bound to protein did not change (96.65% ± 0.29 and 96.81% ± 1.4 respectively). However unbound T levels remained 1/4 to 1/2 that of normal adult females.In 13 patients with primary hypogonadism, total T concentration was similar to that of Turner patients (13.12 ± 5.4 ng/100 ml) so as were the percentage of T bound to proteins and unbound T levels. The physiological implications of free and bound testosterone concentrations in newborns, prepuberal and hypogonadal children are discussed.