X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of host defense that results from mutations in the gene encoding gp91phox, the large subunit of the phagocyte NADPH oxidase flavocytochrome b. In this study, we constructed a recombinant adeno-associated virus-2 (AAV) vector in which the constitutively active promoter from the human elongation factor- 1alpha (EF-1alpha) gene drives expression of the murine gp91phox cDNA, and tested its ability to integrate and express in a human X-CGD myeloid cell line. The nitroblue tetrazolium (NBT) test of NADPH oxidase activity was used to screen transduced cells for vector-mediated expression of recombinant gp91phox. Between 2 - 14% of cells were NBT-positive in the first several weeks after transduction. Clones with NBT-positive cells persisting several months after transduction had integrated vector by Southern blot analyses, with high level reconstitution of NADPH oxidase activity. In some clones, oxidase activity persisted for at least 8 to 14 months. In the majority, however, vector-derived RNA transcripts declined, although integrated rAAV genomes persisted. Decreased transgene expression was not directly correlated with methylation of the provirus. This study indicates that rAAV vectors can be successfully used for stable gene transfer, integration, and expression of recombinant gp91phoxin a human myeloid cell line for at least 8 - 14 months in the absence of any selection. The EF-1alpha promotor, however, was subject to silencing in a high percentage of clones with integrated rAAV, suggesting that alternative promotors may be desirable for achieving long-term expression in myeloid cells.