Gamma-Interferon (IFN-gamma) is a lymphokine produced by T lymphocytes. We find that recombinant human IFN-gamma induces expression of HLA-D antigens on human promyelocytic leukemia cells (HL-60) and enhances expression of HLA-D antigens on normal human monocytes and macrophages. Induction of both HLA-D antigen expression and HLA-D mRNA accumulation occurs within 1 day of exposure of HL-60 to IFN-gamma and is maximal by day 5. Maximal antigen expression occurs in the presence of 100-500 units of IFN-gamma per ml. IFN-gamma induces expression of DR but not DC antigens on HL-60, as confirmed by using four different murine monoclonal antibodies or one rabbit heterologous antibody. RNA blot data show that IFN-gamma-exposed HL-60 cells contain mRNA for DR alpha and DR beta polypeptides but not for DC alpha and DC beta polypeptides, which also suggests that HLA-DR and HLA-DC gene regions can be independently regulated. IFN-gamma induces 20% of HL-60 cells to differentiate to macrophage-like cells. However, IFN-gamma dose-response studies using both HL-60 cells and a nondifferentiation variant of HL-60 cells (HL-60 blast) clearly show that induction of transcription and expression of HLA-D gene products by IFN-gamma can be uncoupled from expression of other monocyte-macrophage characteristics. Further studies show that IFN-gamma enhances expression of HLA-D antigens on normal human monocytes and macrophages. Expression of the HLA-D antigens is necessary for the interaction of macrophages and T lymphocytes; IFN-gamma may play a fundamental role in this interaction.