Adrenarche is the increased adrenal production of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) that occurs during the prepubertal period. To date, the exact mechanism initiating adrenarche is unknown, although many factors have been postulated. In the present study, we examined the hypothesis that alterations in intra-adrenal expression of 3β-hydroxysteroid dehydrogenase (3βHSD) or 21-hydroxylase (CYP21) within the inner reticularis zone leads to the increased production of 19-carbon (C₁₉) steroids. After conversion of cholesterol to pregnenolone, 17α-hydroxylase/17,20-lyase (CYP17) can metabolize pregnenolone through to DHEA. The enzyme 3βHSD competes for substrate with CYP17 and effectively removes steroid precursor from the pathway leading to DHEA. On the other hand, deficiency in CYP21 expression is known to cause excessive production of adrenal C₁₉ steroids, suggesting that CYP21 could play a role in adrenarche. Thus, a decrease in 3βHSD or CYP21 expression would allow substrate to flow toward the synthesis of DHEA. To determine whether adrenarche results from a decreased expression of 3βHSD or CYP21 in the reticularis, immunohistochemical localization of 3βHSD and CYP21 was performed, and staining intensities compared using adrenal glands from children ages 4 months to 4 yr (n = 12), ages 5–7 yr (n = 9), ages 8–13 yr (n = 9), and adults ages 25–56 yr (n = 8). There were no differences in the zonal expression of CYP21. No difference in 3βHSD staining was observed between the glomerulosa and fasciculata from any age group. However, children age 8 yr and older show a significant decrease in 3βHSD expression in reticularis as compared with the fasciculata. No significant difference was noted for 3βHSD levels between the fasciculata and reticularis for children age 7 yr or younger. The level of 3βHSD expression in the reticularis continued to decrease in the adult adrenals examined. These findings suggest that as children mature there is a decreased level of 3βHSD in the adrenal reticularis that may contribute to the increased production of DHEA and DHEAS seen during adrenarche.