Two noncompeting monoclonal antibodies (mAbs) directed to rat interferon-gamma (IFN-gamma) were used in a sensitive two-site enzyme-linked immunosorbent assay (ELISA). This permitted quantitation of rat IFN-gamma in culture medium, and serum and was quicker and more sensitive than the conventional antiviral bioassay. A standard curve was linear between 0.1 (15 pg) and 10 (1,500 pg) U/ml. There was no reaction with rat IFN-alpha or -beta or human IFN-gamma, but mouse IFN-gamma was detected with a lower limit of sensitivity of 300 U/ml. Complement-dependent inactivation of rat IFN-gamma reduced the sensitivity of the ELISA approximately seven-fold but this could be overcome by adding ethylenediamine tetraacetic acid (EDTA, 10 mM) or other anticomplementary substances to the serum samples. IFN-gamma activity determined by ELISA and bioassay decreased in parallel upon heat denaturation and pH 2.5 treatment, but proteases caused a relatively greater reduction in biological activity.