Monocytes treated with 500 IU/ml human recombinant interferon-gamma (rIFN-gamma) 1 day before and continuously after human immunodeficiency virus (HIV) infection showed no evidence of virus replication 7 days after addition of the viral inoculum. There was no HIV-associated cytopathic effect, no reverse transcriptase (RT) activity or p24 detected in culture fluids, and no HIV RNA or DNA in cell lysates. Furthermore, no evidence of HIV infection was evident in replicate cultures in which all IFN-gamma was removed at 7 days and the cells were cultured for an additional 3 weeks without IFN-gamma. The 50% inhibitory dose for reduction of maximum RT activity in HIV-infected monocyte cultures was about 1 IU/ml IFN-gamma. No increase in HIV replication was evident in monocytes treated with IFN-gamma at any concentration (0 to 5000 IU/ml) or at any time (7 days before to 10 days after HIV infection). In side-by-side experiments with identical monocytes and HIV-1 stock, rIFN-gamma was 10 to 20 times more effective than rIFN-alpha 2b for induction of antiviral activity. With both interferons, significant antiviral activity was evident with monocytes treated 1 day before, at the time of, or up to 3 days after infection. At 7 to 10 days after infection (a time at which less than 20% of total cells were infected with HIV) addition of even high concentrations of IFN-alpha or IFN-gamma had no effect on virus replication. These data suggest that the principal action of IFN-alpha and IFN-gamma was directed against the fluid-phase virus. Cell-cell spread of infection within the HIV-infected monocyte culture and extent of virus replication in HIV-infected cells were not affected by interferon treatment.