The CYP21 gene, which encodes P450c21, the adrenal steroid 21-hydroxylase needed for glucocorticoid synthesis, lies in the major histocompatibility locus only 2.3 kilobase pairs (kb) downstream from the C4 gene. A 300-base pair (bp) proximal promoter and two upstream regions within C4 are needed for expression of mouse CYP21; the human gene also has a proximal promoter, but upstream elements have not been studied. To search for upstream regulatory elements in human CYP21B, we examined up to 9 kb of 5'-flanking DNA by transient transfection into human adrenal NCI-H295A cells. The 300-bp proximal promoter had substantial activity, but constructs retaining the DNA between -4.6 and -5.6 kb had increased activity, indicating the presence of distal elements. This region does not correspond to the mouse upstream regions, lying further upstream within intron 35 of C4B, which encompasses the previously described "Z promoter." DNase I footprinting located two elements, F1 and F2, lying -186 to -195 bp and -142 to -151 bp upstream from the Z cap site (-4862 to -4871 and -4818 to -4827 bp upstream of the CYP21B cap site). Each element formed a specific DNA-protein complex and conferred orientation-independent expression to a heterologous promoter. Mutations abolished formation of the DNA-protein complexes but only partially decreased expression. We identified a third site, F3, lying at -33 to -42 bp from Z. Competitive gel mobility supershift assays and co-transfection studies with SF-1 produced in vitro indicate F2 and F3 bind SF-1; BLAST searches and Southwestern blotting suggest that NF-W2 may bind F1. These results indicate that the Z promoter is a component of the CYP21 promoter needed to drive its adrenal-specific expression and that CYP21 transcription elements within C4 have kept these two genes linked during evolution.