The NADPH:O₂ oxidoreductase of phagocytic leukocytes is an important enzyme for the bactericidal activity of these cells. Cytochrome b₅₅₈ is a membrane component of this enzyme. In X-linked chronic granulomatous disease (Xb^- CGD) the phagocytes are defective in the β-subunit (gp91-phox) of this cytochrome. We have studied the genetic defect in a group of six X-linked CGD patients characterized by complete or partial loss of cytochrome b₅₅₈ with the use of the polymerase chain reaction. All patients had a different single point mutation in the gp91-phox gene, indicating that the genetic defect in Xb^- CGD is very heterogeneous. In one patient the mutation leads to a premature termination codon. In the other five cases these mutations predict incorporation of a different amino acid. The mutations were with one exception found in the N-terminal half of the protein, suggesting that this part of cytochrome b₅₅₈ is important for the binding of the heme or for formation of a stable complex with p22-phox. Two histidyl residues were found that might be ligands of the heme iron.