The in vitro regulation of adult human monocyte DR antigen expression was studied. Normally about 75% of freshly obtained human peripheral blood monocytes express DR antigens as determined by anti-DR and complement-mediated cytotoxicity assays. DR expression on monocytes in unfractionated peripheral blood mononuclear cell cultures persisted to variable degrees for up to 5 d of incubation. However, when the mononuclear cells were thoroughly depleted of nonadherent cells, cultured monocytes consistently exhibited progressively decreased DR expression over 2-5 d of incubation. Readdition of nonadherent cells to the adherent cell population prevented or delayed this decrease in monocyte DR antigen expression. Thus, monocyte DR expression diminished markedly during in vitro incubation; however, the presence of nonadherent cells somehow interfered with this process. In other experiments, peripheral adherent monocytes, which had been cultured for 2-3 d to reduce their DR expression, could be induced to reexpress DR antigens after 2 d of incubation with unpurified lymphokine-containing culture supernatants, recombinant human interferon-alpha, or recombinant human gamma interferon (IFN-gamma). The reinduction of DR expression on human monocytes by lymphokines was abrogated by an antiserum produced to the synthetic N-terminal amino acids of human IFN-gamma, indicating that IFN-gamma is the active mediator in the lymphokine-containing preparations. Monocytes cultured with lymphokines or recombinant interferons also could initiate a significantly greater mixed lymphocyte response than control monocytes. Thus, IFN-gamma-containing lymphokines and recombinant interferons are required to induce human monocyte DR expression and accessory cell capacity in vitro, since in their absence monocytes become DR antigen-deficient. Finally, incubation of unfractionated human mononuclear cells with anti-human IFN-gamma also promoted the loss of monocyte DR expression. These findings suggest that resting lymphocytes are probably capable of producing sufficient IFN-gamma in vitro to result in the maintenance of the monocyte DR phenotype.