We previously reported that serum sex hormone-binding globulin (SHBG) decreases and serum non-SHBG-bound testosterone (T) increases with age in normal prepubertal boys from infancy to late prepuberty. In this study we measured serum SHBG, T, estradiol (E2), and dehydroepiandrosterone sulfate (DS), and we calculated serum non-SHBG-bound T and E2 and free T and E2 in 22 normal prepubertal girls, aged 1-9.4 yr. The girls were divided into 3 groups of different ages: group A, 1.7 +/- 1.0 (mean +/- SE) yr; group B, 4.6 +/- 0.8 yr; and group C, 7.3 +/- 0.8 yr. In group C, mean serum SHBG level was lower, and serum T, non-SHBG-bound T, free T, DS, total E2, non-SHBG-bound E2, and free E2 were higher than in group A or B. Furthermore, a negative correlation was found between serum SHBG and age [y(nmol/L) = 144 - 9.07 x (yr); r = 0.57; P less than 0.001], while positive correlations were found between non-SHBG-bound T and age [y(nmol/L = 0.043 + 0.023 x (yr); r = 0.68; P less than 0.001], non-SHBG-bound E2 and age [y(pmol/L) = 0.69 + 2.82 x (yr); r = 0.6; P less than 0.001], and DS and age [y(nmol/L) = 25.2 + 63.8 x (yr); r = 0.59, P less than 0.001]. In a group of 19 normal age-matched prepubertal boys, we also found a significant correlation between non-SHBG-bound E2 and age. Since non-SHBG-bound sex hormone levels are good indicators of tissue available sex hormones, we conclude that in prepubertal girls, there is a progressive increase in the exposure of peripheral tissues to T and E2 with advancing age. Since sex hormones enhance tissue maturation, these increments might play a role in the somatic and psychic development of girls before the onset of the clinical signs of puberty.