We investigated the NADPH oxidase activity, cytochrome b558 content, and gene expression of gp91-phox and p47-phox in normal Epstein-Barr-virus (EBV)-transformed B lymphocytes, compared to EBV-transformed B lymphocytes from patients with X-linked chronic granulomatous disease (CGD), normal peripheral blood neutrophils or mononuclear cells, and the A301 or C8166 lymphoblastoid cell lines. CGD phenotypes included both "classic" disease with no detectable gp91-phox protein (termed X91(0)) and "variant" phenotype with reduced but detectable gp91-phox protein (X91(-)). Normal EBV-transformed B lymphocytes show a dose-dependent PMA-induced superoxide release. Culturing these cells with IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml), alone or in combination for 7 days, caused a modest increase in their NADPH oxidase activity (P > 0.05 in all situations). Normal EBV-transformed B lymphocytes have lower NADPH oxidase activity and cytochrome b558 content than peripheral blood neutrophils or mononuclear cells (P andlt; 0.05 in all situations). In contrast, they have higher NADPH oxidase activity and cytochrome b558 content than X91(-) CGD EBV-transformed B lymphocytes (P andlt; 0.05 in all situations). A301 or C8166 lymphoblastoid cell lines and X91(0) CGD EBV-transformed B lymphocytes have barely detectable NADPH oxidase activity or cytochrome b558 content (P andlt; 0.05 in all situations). Gene expression studies also show a modest increase in expression and transcription rates of gp91-phox and p47-phox genes in normal EBV-transformed B cells cultured with IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml), alone or in combination for 7 days. We conclude that NADPH oxidase activity and cytochrome b558 content correlate with gp91-phox and p47-phox gene expression in EBV-transformed B lymphocytes.