An in vitro method for the generation of effector suppressor cells (Ts3) was developed. By utilizing this protocol, it was possible to investigate both the cellular and genetic requirements for suppressor cell induction. It was determined that populations containing Ts3 cells can be induced after a 4-day culture of spleen cells and antigen. These Ts3 cells are similar to Ts3 cells generated by in vivo immunization. Both populations are I-J+, bind NP hapten, bind NP hapten, bear receptors which share NPb idiotypic determinants with anti-NP antibodies, function during the effector phase of the immune response, and require activation with Ts2 cells. Generation of Ts3-containing populations required both nylon wool-nonadherent T cells and a nylon-adherent, B cell-enriched population from an Igh-identical donor. T cells cultured with antigen alone or with syngeneic macrophages and antigen did not develop suppressive activity. Lytic treatment of the nylon-adherent population with a B cell-specific monoclonal antibody (J11d) removed the ability to generate suppressor cells. These results imply that the induction of suppressor T cells requires B lymphocytes, and that this induction process is dependent on Igh-linked gene products.