Following a standard immunization protocol with recombinant human interferon-gamma (IFN-gamma), a horse polyclonal antiserum was obtained and evaluated for its ability to interfere with multiple IFN-gamma activities in vitro. Data obtained show that polyclonal horse antiserum neutralizes the antiproliferative activity of IFN-gamma, inhibits the binding of IFN-gamma to cellular receptors, and can up-regulate HLA-DR antigen expression and interfere with its antiviral activity. The broad neutralizing capacity of horse polyclonal antiserum has been assessed on cell lines which differ as to origin and sensitivity to IFN-gamma. Moreover, we observed that this antiserum could inhibit the binding of radiolabeled IFN-gamma to its cellular receptor, its subsequent internalization into the target cell, and its antiviral activity. As it is able to inhibit all the biological activities of IFN-gamma, this antiserum might provide new therapeutic approaches to diseases with evidence of activated cell-mediated immunity.