Studies with animal models have indicated that neutralizing antibodies against human interferon-gamma (HuIFN-gamma) may be used to treat a number of diseases in man. A major handicap for the implementation of this form of therapy is the immunogenicity of antibodies of non-human origin. Antibody fragments that do not contain parts of the most immunogenic regions may help to circumvent this problem. Therefore, we have constructed several antibody fragments [VH (variable fragment of the heavy chain), Fv (variable fragment) and scFv (single-chain Fv)] derived from a murine hybridoma (D9D10) which produces a neutralizing antibody against HuIFN-gamma. cDNAs encoding the variable domains of the L and H chains of D9D10 were cloned by PCR-based techniques in a suitable E. coli expression system. Bacterial clones are described that produce either VH, Fv or scFv. The Ig fragments were secreted into the periplasm and leaked into the culture supernatant. By SDS-PAGE and immunoblot analysis, the fragments were shown to be of the expected size (15, 14 and 30 kDa for VH, Fv and scFv, respectively). Functionality of the recombinant Ig fragments was tested by an ELISA for HuIFN-gamma binding and by a neutralization assay for the antiviral activity of HuIFN-gamma. Fv as well as scFv, but not VH, were found to bind to HuIFN-gamma and to neutralize its antiviral activity. Since it is found that scFv proteins are more stable at physiological temperatures than the Fv, it may have potential usefulness for the treatment of diseases in which overproduction of IFN-gamma plays a crucial role.