The increase in pituitary GH secretion that occurs during mid-late puberty in boys follows an increase in circulating testosterone (T) concentration; the direct mechanism by which this occurs is unknown. We hypothesized that T increases GH secretion during puberty by augmenting hypothalamic output of GHRH. Using constant infusions of a GHRH antagonist, we tested this hypothesis in six early pubertal boys with constitutional delay of growth and adolescence who had a mean chronological age of 14.0 ± 0.3 yr and mean bone age of 11.4 ± 0.2 yr. Blood samples were obtained from subjects every 15 min for 24 h during the overnight infusion of normal saline (2000-0600 h) and again during the overnight infusion of GHRH antagonist (0.33 μg/kg/h) the following night. Subjects then received transdermal T (5-mg patch) for 12 h nightly and were studied again after 4 wk of treatment. Serum samples were assayed for GH and total ghrelin; the percent suppression of GH during GHRH antagonist infusion was calculated. Morning serum T rose from 0.44 ± 0.09 to 4.43 ± 0.74 μg/liter (P = 0.005). T treatment was associated with a 92.6% increase in mean nocturnal GH secretion area under the curve (830 ± 177 to 1599 ± 340 μg/24 h·liter). Infusion of GHRH-antagonist suppressed mean nocturnal GH area under the curve by 29.1% before T treatment (830 ± 177 to 621 ± 168 μg/24 h·liter), and by 29.4% after T treatment (1599 ± 340 to 1182 ± 249 μg/24 h·liter; P = 0.99). Somatotroph sensitivity to GHRH was tested with 0.1- and 1.0-μg/kg doses of GHRH-44 iv; GH response did not change with regard to T treatment. The mean 24-h concentration of total ghrelin was unchanged with regard to T treatment. In summary, nightly transdermal T administration in six boys with constitutional delay of growth and adolescence increased GH output almost 2-fold, whereas the degree of GH suppressibility by GHRH antagonist remained unchanged. We conclude that the T-associated augmentation of GH secretion during early puberty in boys is unlikely to involve an absolute increase in hypothalamic GHRH output.