2‐5A‐dependent RNase is the terminal factor in the interferon‐regulated 2‐5A system thought to function in both the molecular mechanism of interferon action and in the general control of RNA stability. However, direct evidence for specific functions of 2‐5A‐dependent RNase has been generally lacking. Therefore, we developed a strategy to block the 2‐5A system using a truncated form of 2‐5A‐dependent RNase which retains 2‐5A binding activity while lacking RNase activity. When the truncated RNase was stably expressed to high levels in murine cells, it prevented specific rRNA cleavage in response to 2‐5A transfection and the cells were unresponsive to the antiviral activity of interferon alpha/beta for encephalomyocarditis virus. Remarkably, cells expressing the truncated RNase were also resistant to the antiproliferative activity of interferon. The truncated RNase is a dominant negative mutant that binds 2‐5A and that may interfere with normal protein‐protein interactions through nine ankyrin‐like repeats.