Withania somnifera (WS) is an important herb with known antiinflammatory activity. Its molecular mechanism of action has not been investigated. The effect of a WS crude ethanol extract was studied on peripheral blood mononuclear cells of normal individuals and rheumatoid arthritis (RA) patients and synovial fluid mononuclear cells of RA patients in vitro. The WS extract significantly suppressed lipopolysaccharide (LPS) induced production of proinflammatory cytokines TNF-alpha, IL-1beta and IL-12p40 in normal individuals and RA patients, but had no effect on IL-6 production at the protein and transcript level. WS also suppressed LPS activated nitric oxide production in the mouse macrophage cell line, RAW 264.7. The extract inhibited nuclear translocation of the transcription factors NF-kappaB and AP-1 and phosphorylation of IkappaBalpha in normal and RA patients' mononuclear cells. HPLC analysis of the crude extract showed the presence of withaferin A and pure withaferin A also inhibited NF-kappaB translocation. The study demonstrated that the WS crude ethanol extract suppressed the production of proinflammatory molecules in vitro. This activity is partly through the inhibition of transcription factors NF-kappaB and AP-1 by the constituent withanolide. The role of additional constituents needs to be studied. Studies on the mechanism of action of the extract may yield potentially useful compounds for the treatment of inflammatory diseases.