During the screening of suppressor T cell (Ts) hybridomas for antigen-nonspecific suppressive activity, we isolated a strain of Mycoplasma arginini which inhibits B cell antibody production in vitro. The addition of mycoplasma-containing Ts hybridoma culture supernatant to splenic B cells responding to sheep red blood cells (SRBC) and T cell-replacing factor or to trinitrophenyl-lipopolysaccharide (TNP-LPS) suppressed the production of anti-SRBC and anti-TNP plaque-forming cells in a dose-dependent and antigen-nonspecific manner. Inhibition occurred due to the noncytotoxic mycoplasmal infection of B cells in culture and required the physical presence of microorganisms. Cell cycle analysis of acridine orange-stained B cells indicated that mycoplasmal infection did not block cell cycle entry and progression of antigen-activated cells. In addition to a suppressive activity, this strain of mycoplasma was selectively mitogenic for B cells. Furthermore, the mycoplasma failed to stimulate or inhibit T cell proliferation. The suppressive and mitogenic activities were selectively absorbed by mitogen-activated B cells but not T cells. These results indicate that this strain of M. arginini mimics the suppressive activity of an antigen-nonspecific Ts factor selective for B cell antibody production.