For MIF production in response to 3 M KCl extracts of tumor, viable and metabolically active macrophages have been shown to be required to interact with the soluble tumor antigens and then come in contact with immune lymphocytes. The Mø-lymphocyte interaction for MIF production was found to be under the control of genes mapping in the IA subregion of the H-2 complex. However, when intact tumor cells were used as antigen, Mø were not required for immune lymphocytes to produce MIF. In addition, the interaction of immune lymphocytes with tumor cells for MIF production did not require H-2 compatibility. These and other observations strongly suggest that there are two different mechanisms for MIF production and that these may be mediated by two separate subpopulations of immune lymphocytes.