The X-linked form of chronic granulomatous disease (X-CGD), an inherited deficiency of the respiratory burst oxidase, results from mutations in the X- linked gene for gp91(phox), the larger subunit of the oxidase cytochrome b. The goal of this study was to evaluate the impact of retroviral-mediated gene transfer of gp91(phox) on host defense against Aspergillus fumigatus in a murine model of X-CGD. Retrovirus vectors constructed using the murine stem cell virus (MSCV) backbone were used for gene transfer of the gp91(phox) cDNA into murine X-CGD bone marrow cells. Transduced calls were transplanted into lethally irradiated syngeneic X-CGD mice. After hematologic recovery, superoxide production, as monitored by the nitroblue tetrazolium (NBT) test, was detected in up to ≃480% of peripheral blood neutrophils for at least 28 to 35 weeks after transplantation. Neutropbil expression of recombinant gp91(phox) and superoxide production were significantly less than wild-type neutrophils. However, 9 of 9 mice with ≃50% to 80% NBT1 neutrophils after gene transfer did not develop lung disease after respiratory challenge with 150 to 500 A fumigatus spores, doses that produced disease in 16 of 16 control X-CGD mice. In X-CGD mice transplanted with mixtures of wild-type and X-CGD bona marrow, ≤5% wild-type neutrophils were required for protection against A fumigatus challenge. These data suggest that expression of even low levels of recombinant gp91(phox) can substantially improve phagocyte function in X-CGD, although correction of very small percentage of phagocytes may not be sufficient for protection against A fumigatus.