The polyclonal response of murine spleen cells to cell wall lysate (CWL) fractions and purified serotype carbohydrate of Streptococcus mutans has been determined by measuring plaque-forming cell (PFC) responses to trinitrophenyl (TNP), sheep (SRBC), and horse (HRBC) erythrocytes and total IgM synthesis. The immunogenicity of TNP-haptenated cell wall carbohydrate in BALB/c and C3H/HeJ mice in vivo and in spleen cell cultures in vitro has also been evaluated. Spleen cell cultures from both C3H/HeJ and BALB/c mice gave good polyclonal responses to M1 c, M1 g, and other cell wall preparations, as manifested by anti-TNP, -SRBC, and -HRBC PFC responses. Polyclonal responses, which were detectable within 2 days of culture, were highest after 3 days of stimulation. The response pattern obtained with an optimal concentration of these S. mutans cell wall preparations was similar to that seen with the B cell polyclonal activator, LPS. Furthermore, good IgM synthesis was induced in 7-day spleen cell cultures incubated with concentrations of S. mutans cell wall carbohydrate that were similar to those used to induce polyclonal PFC responses. Optimal anti-TNP PFC responses to both TNP-M1 c and TNP-M1 g occurred on day 4 after immunization in BALB/c and C3H/HeJ mice when they were given a single i.p. injection of 10 μg of antigen. Good anti-TNP PFC responses were observed when BALB/c nude (nu/nu) and heterozygous (nu/+) mice were immunized with either TNP-M1 c or TNP-M1 g, suggesting that these antigens are thymus-independent. Furthermore, spleen cell cultures from nu/nu mice gave somewhat higher responses to TNP-carbohydrate antigen than did their nu/+ counterparts. Purified B cells from spleens of BALB/c mice also gave good anti-TNP PFC responses. These findings demonstrate that the cell wall serotype carbohydrate of S. mutans is a polyclonal B cell activator and, when haptenated with TNP, is a thymus-independent antigen.