Chronic granulomatous disease (CGD) is associated with significant morbidity and mortality from infection. The first CGD gene therapy trial resulted in only short-term marking of 0.01% to 0.1% of neutrophils. A recent study, using busulfan conditioning and an SFFV retrovirus vector, achieved more than 20% marking in 2 patients with X-linked CGD. However, oxidase correction per marked neutrophil was less than normal and not sustained. Despite this, patients clearly benefited in that severe infections resolved. As such, we initiated a gene therapy trial for X-CGD to treat severe infections unresponsive to conventional therapy. We treated 3 adult patients using busulfan conditioning and an MFGS retroviral vector encoding gp91^phox, achieving early marking of 26%, 5%, and 4% of neutrophils, respectively, with sustained long-term marking of 1.1% and 0.03% of neutrophils in 2 of the patients. Gene-marked neutrophils have sustained full correction of oxidase activity for 34 and 11 months, respectively, with full or partial resolution of infection in those 2 patients. Gene marking is polyclonal with no clonal dominance. We conclude that busulfan conditioning together with an MFGS vector is capable of achieving long-term correction of neutrophil oxidase function sufficient to provide benefit in management of severe infection. This study was registered at www.clinicaltrials.gov as #NCT00394316.