A mitogenic component, designated fraction C (Fr C), has been purified from a mutanolysin enzyme digest of Actinomyces cell walls by CM Sephadex C-25 ion-exchange and G-100 gel filtration chromatography. Good mitogenic responses were obtained with Fr C over a broad dose range with peak mitogenesis seen with 500 micrograms/culture. Fraction C (mol. wt. = 35,000-40,000) consists of 75% carbohydrate and 23% protein, is non-dialysable, resistant to heat, lysozyme or protease treatment, and partially sensitive to base, and all mitogenic activity is destroyed by either periodate or acid treatment. Fraction C is a B-cell mitogen since it induced responses in nude (nu/nu) and nu/+ BALB/c spleen cell cultures and purified splenic B-cell cultures, but did not stimulate purified splenic T-cell cultures. Similar mitogenic fractions for B cells have been obtained from cell walls of A. naeslundii and from a human isolate of A. viscosus. Good polyclonal IgM synthesis and plaque-forming cell responses to hapten or erythrocytes were obtained in vitro with the purified cell wall fractions derived from all three Actinomyces strains studied. These results indicate that the Actinomyces cell wall possesses a carbohydrate-rich component which activates B cells and may represent a common determinant of this genus.