Alveolar macrophages (AM) from normal rats had immunosuppressive activity to mitogen-induced proliferative responses of splenic lymphocytes. We studied the mechanism and the implication of the nitric oxide synthetase pathway in AM-mediated suppression of concanavalin A (Con A)-induced lymphocyte proliferation. The culture supernatant from AM cultures alone did not have immunosuppressive activity to Con A-induced proliferative responses of non-adherent spleen cells (n-ad SC), but the culture supernatant from co-culture of AM and autologous n-ad SC had this activity. Con A-pulsed AM also liberated the immunosuppressive factor. When AM and autologous n-ad SC were cultured separately under the condition that medium could freely communicate, the culture supernatant did not suppress the Con A-induced proliferative response of n-ad SC. This indicated that the immunosuppressive factor was liberated when AM was activated by cell-to-cell contact with n-ad SC. Further, we examined the immunosuppressive activity of the culture supernatant of co-culture of AM and autologous n-ad SC to Con A-induced responses of allogeneic n-ad SC and xenogeneic murine n-ad SC, and allogeneic mixed leucocyte reaction, and found that this culture supernatant could suppress all these proliferative responses. Nitrate (NO2-) synthesis was markedly augmented in the culture supernatants of Con A-pulsed AM and co-culture of AM and n-ad SC. NG-monomethyl-L-arginine (MMA), a specific competitive inhibitor of the nitric oxide synthetase pathway (NOSP), extinguished both NO2- synthesis by AM and AM-mediated immunosuppressive activity. These data suggest that NOSP was important in AM-mediated suppression of Con A-induced lymphocyte proliferation.