Human 2'-5' oligoadenylate synthetases (OAS) are interferon inducible proteins which， upon activation by double-stranded RNA， polymerize ATP into 2'-5' linked oligoadenylates. Here， we probe the RNA cofactor specificity of the two smallest isozymes， OAS1 and OAS2. First， we demonstrate the purification of recombinant OAS2 from human cells and quantified enzymatic activity relative to OAS1. We then confirmed that both OAS2 domains， as opposed to only the domain containing the canonical catalytic aspartic acid triad， are required for enzymatic activity. Enzyme kinetics of both OAS1/OAS2 in the presence of RNA binding partners enabled characterization of the maximum reaction velocity and apparent RNA-protein affinity of activating RNAs. While OAS1 can be activated by dsRNA greater than 19bp， OAS2 showed a marked increase in activity with increasing dsRNA length with a minimum requirement of 35bp. Interestingly， activation of OAS2 was also more efficient when the dsRNA contained 3'-overhangs despite no significant impact on binding affinity. Highly structured viral RNAs that are established OAS1 activators were not able to activate OAS2 enzymatic activity based on the lack of extended stretches of dsRNA of greater than 35 bp. Together these results may highlight distinct subsets of biological RNAs to which different human OAS isozymes respond.