The existence of three distinct forms of 2-5A synthetase, p40-p46, p69, and p100, has been established in interferon-treated human cells. The expression of these enzymes varies according to the cell type studied, and their properties are relatively different. By the use of polyclonal antibodies specific to p69, we have cloned several cDNAs which identify four interferon-induced RNAs of 5.7, 4.5, 3.7, and 3.2 kilobases (kb). Analysis of the nucleotide sequence of three full-length cDNAs (5.6, 3.1, and 2.9 kb) revealed that they have a common open reading frame of 683 amino acids with different 3' termini; cDNAs 5.6 and 3.1 have an extension of 4 amino acids, whereas cDNA 2.9 has an extension of 44 amino acids. In vitro transcription-translation of cDNAs 3.1 and 2.9 kb generated proteins of 69 and 71 kDa, respectively. Both proteins bind a monoclonal antibody specific for p69 and can synthesize 2-5A. The deduced amino acid sequence of p69 revealed that it can be divided into two homologous and adjacent domains, each sharing strong homology to the first 346 amino acids common to the two isoforms of the small 2-5A synthetase (p40, p46). These results suggest that p69 might have two functional catalytic domains required for 2-5A synthetase activity and favor the hypothesis that its gene might have derived from the fusion of two ancestral genes analogous to the small 2-5A synthetase gene.