Regulatory effects of antibodies to cell-surface antigens have been examined in an in vitro clonal assay for B lymphocytes. In this system, cell contact is prevented by a semisolid gel matrix, and many of the factors affecting cell proliferation have been described. Inclusion of antibodies to mu, kappa, or Ia in these cultures reduced the size and number of proliferating foci. As little as 1 microgram/ml of anti-mu antibody prevented formation of colony-size aggregates. Pretreatment with these antibodies before culture followed by wash had no effect. Divalent F(ab')2 anti-IgM antibodies were as effective as intact antibodies, but monovalent Fab fragments were not inhibitory. Anti-gamma1 antibodies, anti-alpha antibodies, or cell-bound antigen-antibody complexes had no effect on clonal proliferation. These findings suggest that a negative signal can be delivered to B cells during a critical period of their activation via certain surface receptors. Modifications in culture conditions result in the selective propagation of different B-cell subpopulations.