Abstract We have generated polyclonal antisera and monoclonal antibodies against recombinant guinea pig IFN-γ. These antibodies were used to inhibit the function of IFN-γ in vitro and to establish a capture ELISA system for the detection and quantitation of this cytokine. Although recombinant protein expressed in E. coli was available in abundance, it was only of limited value to develop a capture ELISA which detects the native cytokine, since only a limited number of monoclonal antibodies reacted both with the recombinant and the native protein. Positive test results in an initial ELISA setup with recombinant IFN-γ were not predictive for the detection of IFN-γ from activated T-lymphocytes in the same assay. After evaluating several different combinations of rabbit antisera and monoclonal antibodies, an assay system was established which uses two mouse monoclonal antibodies as capture and detecting reagents. Three of the monoclonal antibodies and the rabbit antisera were able to block the function of guinea pig IFN-γ when assayed in a luciferase reporter assay.