IL-10, a newly designated cytokine primarily produced by the Th2 subset of CD4+ T lymphocytes and Ly-1+ B lymphocytes, has recently been hypothesized to inhibit cytokine production by Th1 T cell clones by blocking accessory cell- (AC) dependent costimulatory function. To evaluate the effect of IL-10 on Con A-induced proliferative responses of resting murine T cells, purified T cells were cultured with different types of AC. The addition of IL-10 produced a 70 to 90% inhibition of resting T lymphocyte proliferation when purified populations of macrophages were used as AC, but had no effect on the AC function of T-depleted spleen cells, activated B cells, dendritic cells, or L cells. The inhibitory effects of IL-10 were inversely related to the concentration of mitogen and could be reversed by the addition of the neutralizing anti-IL-10 mAb, SXC1. The inhibition of macrophage AC function was not related to the induction of a suppressor cytokine as stimulation by mixtures of macrophages and limiting numbers of dendritic cells was not inhibited. The decrease in proliferative responses was primarily secondary to inhibition of IL-2 production although the failure of exogenous IL-2 to completely reconstitute the response suggested that IL-10 may also exert inhibitory effects on the induction of expression of a functional IL-2R. These results are most consistent with a model in which IL-10 inhibits the induction of expression on macrophages of a critical costimulatory molecule that may be constitutively expressed on other types of AC.