A new, specific and sensitive one-site ELISA for precise quantification of human interferon-gamma (HuIFN-gamma) at low levels in 50% human serum samples has been developed. The assay is based on the assumption that biologically active HuIFN-gamma is present exclusively as a dimer. Thus, in contrast to previous reports, the ELISA is based on a single monoclonal antibody (MAb) which is used in two ways: as "catching" antibody and as HRPO-labelled conjugate. The sensitivity could be improved five-fold by addition of (NH4)2SO4 to the conjugate solution; the lowest detectable amount of HuIFN-gamma is < 0.5 mu/ml. Non-specific interactions were not seen in interferon samples taken from cultures, or samples diluted in ordinary media or 1% BSA. However, > 30% of the (serum) samples gave non-specific false-positive results when the method was applied to samples containing 50-100% human serum from different donors. The false signals were related to the donors but could-at the expense of the sensitivity which was reduced to 1 mu/ml-be abolished by PEG treatment of the (donor) serum samples.