Three single-chain antibody fragments that recognize the extracellular human interferon γ receptor α-chain (IFNγR), and inhibit the binding of human IFNγ, have been produced in Escherichia coli. These fragments are derived from murine anti-receptor monoclonal antibodies, and comprise the variable heavy (V_H) domain linked to the variable light (V_L) chain through a 15 amino acid linker [(GGGGS)₃]. Using surface plasmon resonance technology (BIAcore), the soluble proteins were shown to retain a high affinity for recombinant IFNγR, and by radioimmunoassay to possess high inhibitory activity towards IFNγ-binding to human Raji cells. The antibody fragments most likely recognize epitopes that overlap the cytokine binding site on the receptor surface. Attempts to dissect further the antibodies to isolated V_H- and V_L-chains and to synthetic linear and cyclic peptides derived from the individual complementarity determining regions failed to afford fragments with significant IFNγR binding affinity. Nevertheless, these native-like variable region fragments and petidomimetics derived from them are of interest in the design of novel IFNγR antagonists.