The canine interferon-gamma (cIFN-gamma) chromosomal gene was isolated from a recombinant library, containing the entire dog genome, by screening with a rat IFN-gamma genomic probe. The structural organization of the cIFN-gamma gene closely resembles that of the human, murine, and rat IFN-gamma genes. It contains three intervening sequences and encodes a signal sequence of 23 amino acids followed by a mature protein of 143 amino acids. Two potential N-glycosylation sites are located at positions 16 and 83 of the mature protein. Comparison of the cIFN-gamma protein sequence with that of the corresponding murine, rat, human, and bovine proteins revealed a homology of 40%, 42%, 65%, and 76%, respectively. The cIFN-gamma gene was expressed under control of the simian virus 40 early promoter in Chinese hamster ovary (CHO) cells, deficient in dihydrofolate reductase (DHFR), after cotransformation with a plasmid containing the murine dhfr gene. Initial transformants with dhfr+ phenotype produced cIFN-gamma titers ranging from 10 to 10,000 laboratory units (LU)/ml of culture medium. A cIFN-gamma cDNA sequence was identified in a cDNA library constructed with partially purified RNA from a cIFN-gamma-producing CHO cell line. In vitro transcription of the cDNA and translation of the mRNA in Xenopus laevis oocytes resulted in secreted IFN-gamma with an antiviral activity specific for canine cells.