2',5'-Oligoadenylate [2-5(A)] synthetases are a family of interferon-induced enzymes that polymerize ATP into 2'-5'-linked oligoadenylates in the presence of double-stranded RNA (dsRNA), their cofactor. The 2-5(A) molecules, in turn, activate the latent ribonuclease RNase L by promoting its dimerization. The 2-5(A) synthetase pathway has been implicated in interferon's antiviral and anticellular activities. In addition to their interesting cellular properties, these enzymes are also enzymologically interesting because they are the only known template and primer independent nucleotide (DNA or RNA)polymerases that synthesize 2'-5'-linked oligonucleotides. Moreover, their mode of activation by dsRNA remains unknown. In the past, biochemical and structure-function studies have been hampered by the lack of a convenient system for expressing recombinant 2-5(A) synthetases. These proteins are toxic to mammalian cells, probably because of RNase L activation, and proteins produced in bacteria do not have full enzymatic activity. To circumvent these problems, we have developed a baculovirus-insect cell system for high-yield expression of the small and medium isozymes. Here, methods are described for the production, purification, and characterization of the mouse small (9-2) (S. K. Ghosh, J. Kusari, S. K. Bandyopadhyay, H. Samanta, R. Kumar, and G. C. Sen, 1991, J. Biol. Chem. 266, 15293-15299) and human medium (P69) (I. Marie and A. G. Hovanessian, 1992, J. Biol. Chem. 267, 9933-9939) 2-5(A) synthetase isozymes and their mutants using the insect cell system. We also report methods for studying 2-5(A) synthetase-dsRNA interactions and protein-protein interactions among the subunits of the two isozymes.