Bupleuri Radix is a commonly used medicinal plant in Kampo medicine, and its hot water extracts show mitogenic activity to murine lymphocytes. In this paper the mitogenic substances in the hot water extracts of Bupleuri Radix (Bup-HWE) were fractionated and characterized physicochemically and immunologically. Most of these substances were recovered from mol. wt of more than 200 kDa fraction (fr. C-13). Separation of fr. C-13 by phenol-water fractionation method gave water soluble and phenol soluble mitogenic substances. These substances showed the activity even in C3H/HeJ mice, and polymyxin B or lysozyme treatment did not abrogate the activity, suggesting that the active substances are not related to bacterial lipopolysaccharide. Treatment of the mitogenic substances recovered from the phenol layer with NaClO₂, a polyphenol degrading chemical, significantly reduced the activity, but pronase and pectinase treatments were not effective. The mitogenic substances in the water layer were active even after NaClO₂ treatment. These findings suggested that the mitogenic substances of Bup-HWE are large molecular weight polyphenolic compounds and polysaccharide. The mitogenic substances are suggested to be B cell mitogens.