Interferon-gamma (IFN-gamma) is a cytokine produced by T lymphocytes and Natural Killer cells which has a key function in resistance against infections. Baboon (Papio anubis) IFN-gamma was produced by stimulation of baboon splenocytes with a lysate of Staphylococcus aureus. This interferon was active on human cells and could be seroneutralized with a polyclonal antiserum against human IFN-gamma, but not with antisera against human interferon-alpha and interferon-beta. Poly(A)(+)-RNA was isolated from baboon splenocytes and fractionated according to its sedimentation coefficient by sucrose density centrifugation. BaIFN-gamma mRNA was present in the 15 S fraction as was shown by hybridization with a human IFN-gamma cDNA probe. A cDNA library was constructed and a clone containing the complete BaIFN-gamma cDNA was isolated. The cDNA codes for a polypeptide of 165 amino acids of which the 23 N-terminal may serve as signal peptide. BaIFN-gamma differs at 11 residues from human IFN-gamma. Southern analysis of chromosomal DNA confirmed some of the nucleotide sequence differences between baboon and human IFN-gamma. The baboon IFN-gamma cDNA was placed under control of a trc promoter and brought to expression in Escherichia coli cells. Recombinant baboon IFN-gamma could be seroneutralized with certain monoclonal anti-human IFN-gamma antibodies. The presented work leads to the availability of recombinant baboon IFN-gamma for animal experiments but also yields new insight in the structure-function relationship of IFN-gamma.