The adrenal inner zone antigen (IZA), which reacts specifically with a monoclonal antibody raised against the fasciculata and reticularis zones of the rat adrenal, was previously found to be identical with a protein variously named 25‐Dx and membrane‐associated progesterone receptor. IZA was purified as a glutathione S‐transferase‐fused or His₆‐fused protein, and its molecular properties were studied. The UV‐visible absorption and EPR spectra of the purified protein showed that IZA bound a heme chromophore in high‐spin type. Analysis of the heme indicated that it is of the b type. Site‐directed mutagenesis studies were performed to identify the amino‐acid residues that bind the heme to the protein. The results suggest that two Tyr residues, Tyr107 and Tyr113, and a peptide stretch, D99–K102, were important for anchoring the heme into a hydrophobic pocket. The effect of IZA on the steroid 21‐hydroxylation reaction was investigated in COS‐7 cell expression systems. The results suggest that the coexistence of IZA with CYP21 enhances 21‐hydroxylase activity.