The aim of this work was to analyze the effect of Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on NADPH oxidase activity and gp91-phox gene expression in human colostrum macrophages (CM), peripheral blood monocytes (PBM), and myelomonocytic THP-1 cells. We also investigated the effect of IFN-gamma on the release of TNF-alpha by these cells. Our results show that under basal culture conditions, CM release more superoxide than PBM and THP-1 cells (p andlt; 0.05). The addition of IFN-gamma, alone or in combination with TNF-alpha, increased spontaneous superoxide release by PBM and THP-1 cells (p andlt; 0.05) and increased phorbol myristate acetate (PMA)-stimulated superoxide release by CM, PBM, and THP-1 cells (p andlt; 0.05). The NADPH oxidase activity of THP-1 cells consistently remained lower than that of CM or PBM, despite a dramatic response to IFN-gamma and TNF-alpha. Under basal conditions, gp91-phox gene expression was significantly higher in CM and PBM compared with THP-1 cells (p andlt; 0.05). The addition of IFN-gamma alone or in combination with TNF-alpha caused a dramatic increase in gp91-phox gene expression in THP-1 cells (p andlt; 0.05) but not in CM or PBM. Under basal conditions or in the presence of IFN-gamma, CM released more TNF-alpha than PBM or THP-1 cells (p andlt; 0.05). In addition, PBM released more TNF-gamma than THP-1 cells (p andlt; 0.05). IFN-gamma did not significantly augment the release of TNF-alpha by these cells (p > 0.05). Thus, IFN-gamma and TNF-alpha induced equivalent gp91-phox gene expression in THP-1 cells compared with CM or PBM but did not bring about equivalent NADPH oxidase activity. TNF-alpha release was higher in more mature cells. This partial divergence of gp91- phox gene expression, NADPH oxidase activity, and TNF-alpha release is probably a consequence of different events of myeloid cell biology and relates at least in part to cell differentiation state.