The regulation of androgen synthesis during puberty in females is complicated, with changes in steroidogenic and peripheral interconversion capacity. In the present study we have investigated the diurnal rhythm of testosterone secretion in 56 healthy girls before and during puberty, up to 2 yr postmenarche. The girls' ages ranged between 4.6-16.5 yr, and their height SD scores ranged between -3.6 and +3.7. One to 5 serum profiles (seven samples per 24 h) were taken from each girl for steroid measurements, and a total of 84 serum profiles were obtained. Serum testosterone concentrations were determined using a RIA with a detection limit of 30 pmol/L. The results demonstrate that there is a diurnal rhythm of testosterone secretion during both prepuberty and puberty in girls. The pattern has its nadir in the late evening or just after midnight, with the highest levels in the morning (0600-1000 h). Serum testosterone concentrations in prepubertal girls were significantly lower than those in pubertal girls and were significantly lower in early puberty than in girls in mid- or late puberty. No differences were found in levels between girls in midpuberty or late puberty. Before puberty, serum testosterone concentrations correlated with serum dehydroepiandrosterone sulfate, consistent with the adrenals being the major source of testosterone. After the onset of puberty, a correlation between testosterone and 17beta-estradiol was seen, consistent with the ovaries being the major source of testosterone during puberty. Furthermore, the present study showed that there is a relative hyperandrogenicity in early puberty, with high levels of androgens relative to estrogens.