Ginseng radix, the dried root of Panax ginseng C. A. Meyer, has been shown to enhance the ability to resist microbial infections. Proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, and interferon-gamma (IFN-gamma) are produced by macrophages treated with ginseng radix extract (GRE) in vitro as well as in vivo. However, the molecular mechanisms of the production are still not clear. In the present study, we demonstrated that production of TNF-alpha and IFN-gamma was induced by GRE in spleen cells and peritoneal macrophages from C3H/HeN mice but was impaired in C3H/HeJ mice carrying a defective toll-like receptor-4 (TLR-4) gene. In addition to these cytokines, the expression of IFN-beta and inducible nitric oxide synthase (iNOS) mRNAs was also increased in GRE-treated C3H/HeN spleen cells. We investigated the possibility that GRE contains a lipopolysaccharide (LPS)-like component. However, GRE induced production of TNF-alpha and IFN-gamma in the presence of polymyxin B, an LPS inhibitor. Furthermore, a Limulus amebocyte lysate assay showed that the endotoxin content of GRE was below the threshold level of 1 ng/ml LPS. These results suggest that GRE contains a non-LPS agent that enhances innate immunity through production of proinflammatory cytokines via TLR-4.