The diagnosis of growth hormone (GH) deficiency is strongly influenced by age, body mass index and presence of gonadal steroids. Priming with oral estradiol (E2) is one possible way to overcome the impact of variable levels of sex steroids. We describe the effects of transdermal estradiol (E2-t) priming on GH response after clonidine stimulation in prepubertal children with familial short stature (group 1, n = 12) or constitutional growth delay (group 2, n = 22). All patients underwent a clonidine test (0.1 mg/m2, p.o.) followed by a clonidine plus E2-t test (50 microg/day) with a 7-day interval. Before E2-t, basal GH and insulin-like growth factor-I (IGF-I) values were similar in the two groups. After E2-t priming, basal GH was significantly higher only in group 2. When compared with group 1, patients from group 2 had a significant increase of GH peak response when submitted to E2-t. The number of patients in both groups with adequate GH peak response was higher after E2-t priming. We conclude that E2-t priming is able to increase GH peak response after clonidine stimulation and also improves the accuracy of the clonidine test in the diagnosis of GH deficiency. Compared to oral administration, E2-t delivery can prevent liver toxicity, providing a more physiological mechanism of GH secretion.