Abstract We examined the effect of recombinant human macrophage-colony stimulating factor (rhM-CSF) on mouse macrophage accessory functions in vitro.The addition of rhM-CSF to an allogenic mixed lymphocyte culture (MLC) consisting of mouse spleen cells suppressed the proliferation of lymphocytes in a concentration-dependent manner. However, rhM-CSF did not suppress the mixed lymphocyte reaction (MLR) on the MLC that contained only purified T and dendritic cells. This suggested that the suppressive effect of rhM-CSF on the MLR occurs via spleen macrophages. Suppression of the MLR by rhM-CSF could not be neutralized by the addition of synthetic inhibitors of prostaglandin E 2(indomethacin) and nitric oxide ( NG-monomethyl- L-arginine). An antibody specific to mouse interleukin-10 (IL-10), which can neutralize the inhibitory effect of recombinant IL-10 in vitro,did not prevent rhM-CSF-induced suppression of the MLR. Even higher concentrations of IL-1 could not overcome the inhibitory effect of rhM-CSF. Moreover, culture supernatants of macrophages stimulated with rhM-CSF had a suppressive effect on the MLR. The concentrations of transforming growth factor β (TGF-β) and IL-10 in the suppressive culture supernatants were lower than those in the supernatants of nonstimulated macrophages. The supernatants suppressed the proliferation of the LBRM 33 TG6 mouse T cell line, but it did not affect the proliferation of other cell lines (L1210, L5178Y-R, and MC/9). These results suggest that rhM-CSF modulates a macrophage accessory function by stimulating macrophages to secrete a suppressive factor that causes suppression of the T cell response in vitro.