Oldenlandia diffusa (OD) has been used to treat malignant tumors. In this study using mouse peritoneal macrophages, we have examined the mechanism by which OD regulates nitric oxide (NO) production. When OD (1 mg/ml) was used in combination with 10 U/ml of recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO production (36.13+/-7.12 microM) by the Griess method (nitrite). Treatment of macrophages with rIFN-gamma plus OD (1 mg/ml) caused a significant increase in tumor necrosis factor-alpha (TNF-alpha) production (4.49+/-1.43 ng/ml) by enzyme-linked immunosorbent assay. The increased production of NO and TNF-alpha from rIFN-gamma-plus OD-stimulated cells was almost completely inhibited by pretreatment with 100 microM of pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa B (NF-kappaB). PDTC also inhibited phosphorylation of IkappaB in rIFN-gamma-plus OD-stimulated cells. These findings demonstrate that OD increases the production of NO and TNF-alpha by rIFN-gamma-primed macrophages and suggest that NF-kappaB plays a critical role in mediating these effects of OD.