Mouse spleen cells capable of specifically binding intrinsically tritium-labeled polymerized flagellin (POL) (labeling by biosynthesis of flagellar protein) via IgM receptors were found to comprise a distinct population of about 20-50 cells per 10(6) lymphocytes. Evidence is presented that the majority of mouse spleen cells binding tritium-labeled POL undergoes blastogenesis after antigen capping, antigen shedding, and receptor reformation. Under conditions of tolerance induction in vitro, however, loss of antigen from the cell surface was inhibited. Such inhibition of antigen redistribution and shedding was reversed by a short pulse of colchicine and new antigen receptors were formed. In spite of this, colchicine had no effect on the tolerant state. However, tolerance could be broken, regardless of presence or absence of the alkaloid, with radioresistant theta-negative accessory (A) cells (adherent cells) from normal but not from tolerant spleen cell populations. "Tolerant" A cells, although they were incapable of cooperating in a response to POL, were capable of participating in a response to a second unrelated antigen. It is concluded that tolerance to POL in vitro is induced by mechanisms other than the physical blocking of bone marrow-derived (B) cell receptors by antigen. Most likely, the discrimination by the B cell between a tolerogenic and immunogenic signal is mediated by A cells.