The capacity of the trinitrophenyl haptenic group coupled to a series of chemically dissimilar carriers to cross-stimulate putative T-dependent and T-independent B-cell subpopulations was determined by using an in vitro limiting dilution technique to generate primary IgM responses. TNP-Ficoll and TNP-dextran, two T-independent antigens with little or no polyclonal mitogenicity, stimulate the same population of anti-TNP precursors, which is distinct from the precursor population activated by TNP-LPS, a T-independent polyclonal mitogen, or by TNP-HRBC, a T-dependent antigen. TNP-LPS and TNP-HRBC activate the same precursor population, indicating that LPS can substitute for the T cell signal in T-dependent B-cell responses, whereas nonmitogenic T-independent antigens cannot. However, the cumulative evidence from this and other laboratories suggests that LPS and T-dependent antigens activate B cells by different mechanisms. TNP conjugates of Ficoll and dextran, which are relatively poor inducers of polyclonal B cell activation, induced larger anti-TNP clones than did TNP-LPS, a strong polyclonal mitogen. Macrophages are required for the anti-TNP-Ficoll/anti-TNP-dextran response, whereas, a similar requirement has not been shown for the anti-TNP-LPS response. Thus, macrophages may function as polyclonal B cell activators in T-independent responses. Experiments in which TNP was coupled directly onto the macrophage surface support this hypothesis. B-cell heterogenity in T-dependent responses is suggested by experiments using the C3 receptor as a marker for functional subpopulations of B cells. Murine T cells cooperate with B cells that carry a receptor for C3 and with at least some B cells which lack the C3 receptor in a primary in vitro antibody response. In vitro culture experiments using populations of B cells fractionated on the basis of the C3 receptor showed that CR+ cells were unable to make T-dependent antibody responses in the presence of anti-C3 antibody, whereas the response of CR- B cells was unaffected. Using irradiated, carrier-primed spleen cells from B10.A mice as a source of helper cells for B cells derived from various congenic strains in an in vitro primary IgM response to TNP-KLH, CR+ B cells cooperated across haplotype differences in the I region of the MHC, whereas CR- B cells did not. Preliminary mapping experiments for the genetic restriction of CR- B cells suggest complementation between the I-A and I-C subregions of the MHC. These findings tentatively suggest the existence of alternative cooperative pathways between T cells and B cell subpopulations.