The immediate-early gene NGFI-B encodes an orphan nuclear receptor that binds DNA as a monomer and activates transcription through a canonical response clement (NBRE). NGFI-B is expressed under basal conditions and in response to external stimuli in many mammalian tissues. In particular, NGFI- B expression is dramatically elevated in the adrenal cortex in response to stress and in Y1 adrenocortical cells in response to adrenocorticotropin. NGFI-B activates transcription through an NBRE of the gene encoding 21- hydroxylase (P450(c21)) in Y1 cells. Steroidogenic factor 1 (SF-1), a homolog of NGFI-B, also activates the P450(c21) promoter. To examine the influence of these factors on P450(c21) expression in vivo and the function of the hypothalamic-pituitary-adrenocortical axis as a whole, we generated NGFI-B (- /-) mice. These mice thrive and reproduce normally and maintain normal basal adrenocorticotropin, corticosterone, and P450(c21) mRNA levels. In response to increases in adrenocorticotropin, NGFI-B (-/-) and wild-type mice demonstrated equivalent increases in serum corticosterone levels. Furthermore, and in contrast to in vitro results, no increases in P450(c21) mRNA levels were observed in response to increases in adrenocorticotropin in NGFI-B (-/-) or wild-type mice. While SF-1 mRNA levels were not increased with increased steroidogenic demand, adrenal expression of Nurr1, a dose homolog of NGFI-B, was induced to a greater extent by lipopolysaccharide in NGFI-B (-/-) mice than in wild-type mice. Finally, when the administration of dexamethasone for suppression was stopped, P450(c21) mRNA and serum corticosterone levels recovered at the same rate in wild-type and NGFI-B (- /-) mice. Thus, while NGFI-B appears poised to affect the structure and function of the adrenal gland, the gland functions normally in its absence, suggesting that other factors, including Nurr1 and SF-1, are sufficient to drive P450(c21) expression in mice and maintain normal steroidogenesis.