We show that APS, a polysaccharide isolated from the roots of Astragalus membranaceus, significantly induces nitric oxide (NO) production and inducible NO synthase (iNOS) transcription through the activation of nuclear factor-kappaB/Rel (NF-kappaB/Rel). In vivo administration of APS induced NO production by peritoneal macrophages of B6C3F1 mice. APS also dose-dependently induced the production of NO in isolated mouse peritoneal macrophages and RAW 264.7, a mouse macrophage-like cell line. Moreover, iNOS protein and mRNA transcription were strongly induced by APS in RAW 264.7 cells. To further investigate the mechanism responsible for the induction of iNOS gene expression, we investigated the effect of APS on the activation of transcription factors including NF-kappaB/Rel and Oct, whose binding sites were located in the promoter of iNOS gene. Treatment of RAW 264.7 cells with APS produced strong induction of NF-kappaB/Rel-dependent reporter gene expression, whereas Oct-dependent gene expression was not affected by APS. Nuclear translocation and DNA binding activity of NF-kappaB/Rel was significantly induced by APS. The treatment with NF-kappaB SN50, an inhibitor of NF-kappaB/Rel nuclear translocation, effectively inhibited the activation of NF-kappaB/Rel binding complexes and NO production. In conclusion, we demonstrate that APS stimulates macrophages to express iNOS gene through the activation of NF-kappaB/Rel.