2-5Adependent RNAase, an interferon-induced enzyme that is activated by 5’-phosphorylated, 2’, 5’-linked oligoadenylates (2-5A), is implicated in both the molecular mechanisms of interferon action and the fundamental control of RNA stability in mammalian cells. Here we report the expression cloning and analysis of murlne and human 2-SA-dependent RNAases. The 2-5A binding properties and RNAase activities of recombinant and naturally occurring forms of 2-SA-dependent RNAase were identical. Interferon induction of 2-5A-dependent RNAase expression was demonstrated by measuring the mRNA levels in cells treated with interferon and cycloheximide. Analysis of aligned murlne and human SdA-dependent RNAase sequences revealed several intriguing features, including similarity to RNAase E, which is implicated in the control of mRNA stability in E. COIL Interestingly, a duplicated phosphate-binding loop motif was determined by deletion analysis and site-directed mutagenesis to fUnC-tion in the binding of 2-5A.