3β-Hydroxysteroid dehydrogenase type 2 (HSD3B2) is a steroid-metabolizing enzyme that is essential for adrenal production of mineralocorticoids and glucocorticoids. Thus, HSD3B2 is expressed at high levels in the glomerulosa and fasciculata, where these steroids are produced. In contrast, the production of dehydroepiandrosterone (DHEA) and DHEA sulfate in the adrenal reticularis is inversely correlated with the expression of HSD3B2. The reasons for the zonal expression of HSD3B2 are not known but represent an important aspect in the biochemical zonation of the adrenal. Using microarray, real time reverse transcriptase-PCR, immunohistochemistry, and HSD3B2 promoter analysis, we demonstrate that the NGFIB family of nuclear hormone receptors plays a critical part in the regulation of HSD3B2 transcription and may play an important role in the functional zonation of the adrenal gland. Microarray analysis of cortisol- versus DHEA sulfate-producing adrenal tissue demonstrated that NGIFB paralleled expression of HSD3B2 with expression much higher in cortisol-producing adrenal tissue; this observation was also demonstrated using real time reverse transcriptase-PCR analysis. In addition, immunohistochemistry confirmed that within adult and fetal adrenal gland NGFIB expression paralleled expression of HSD3B2. Transient transfections into H295R adrenal cells demonstrated that NGFIB family members enhanced HSD3B2 reporter activity but had no effect on a 17α-hydroxylase (CYP17) promoter construct. Deletion and mutational analyses of the 5′-flanking region of the HSD3B2 gene identified a consensus NGFIB response element that bound NGFIB in mobility shift assays. Infection of cultured human adrenal cells with adenovirus-containing NGFIB increased cortisol production by 8-fold and increased expression of HSD3B2 mRNA 26-fold over that observed in mock-infected cells. In primary cultures of adrenal cells, ACTH, an activator of HSD3B2, rapidly induced expression of NGFIB. These results suggest that NGFIB plays a crucial role in adrenal zonation by regulating HSD3B2 gene transcription.