We have already demonstrated that various activities including NO (nitric oxide) synthesis in vivo and in vitro significantly differ between triple helical (SPG) and single helical (alkaline-treated SPG, SPG-OH) beta-glucans. It was previously suggested that the single helical conformer of beta-glucan (SPG-OH) was dominant in cytokine production and subsequent NO synthesis in vitro. In this study, we analyzed production of inducible nitric oxide synthase (iNOS) induced by beta-glucans in vitro and in vivo. The iNOS production was enhanced in proteose peptone-induced peritoneal macrophages (PMs) cultured with SPG-OH in the presence of IFN-gamma for 24 h, and SPG-OH-induced PMs. Moreover, SPG-OH was effective for iNOS production not only in isolated macrophages but also in tissue macrophages, whereas SPG was less effective. These findings suggest that a single helical conformer is essential for iNOS production, and that NO synthesis by beta-glucans is closely related to iNOS production.