W e recently reported the biological activity and some of the biochemical characteristics of a factor produced by a human T cell hybrid clone able to block hematopoietic progenitor cell proliferation. This 85-kD protein factor, which we have termed colony-inhibiting lymphokine (CIL), has growth regulatory activity on bone marrow precursors bearing Ia (class II) antigens of either granulocytic-monocytic (CFU-GM) or erythroid lineage (BFU-E and CFU-E). Experiments aimed to investigate the specificity of the inhibitory effect on hematopoietic progenitor cell growth suggested that the expression of HLA-DR surface antigens was required on the target cells. We describe in this communication how DR+ cell lines ceased dividing after a few days of culture in the presence of CIL, whereas DR- cell lines were completely unaffected. The increased DR expression on the ML3 cell surface, mediated by the activity of the gamma interferon (IFN gamma), increases the sensitivity to the growth inhibition factor of the ML3 cell line. To verify the hypothesis that the DR antigens might serve as receptors for the factor, enabling it also to interfere in the immune response, we tested CIL in a mixed lymphocyte reaction (MLR), one of the best known in vitro Ia antigen-dependent T cell-mediated immune responses. CIL is able to block major histocompatibility complex-allogeneic MLR both in human and mouse systems. The data indicate that CIL recognizes a nonpolymorphic structure (presumably on all Ia molecules) presented by stimulator cells of either species, and thereby interferes with specific interactions between stimulator and responder cells. Blocking of the alloantigen stimulation stage is also indicated, since CIL is effective only if added to the culture medium during the first 48 h of the MLR. Finally, mouse monoclonal anti-DR antibodies are able to sharply reduce CIL activity on sensitive DR+ cell lines. CIL may act physiologically as a multifunctional mediator in a complex network that links regulation of bone marrow differentiation and the generation of immune responses.