The oligonucleotide pppA2′ p5′ A2′ p5′ A is synthesized by extracts from interferon-treated mouse L cells in the presence of double-stranded RNA. This compound is a potent inhibitor of protein synthesis in cell-free systems prepared from L cells or rabbit reticulocytes. After an initial lag, rates of protein synthesis in vitro are severely depressed in the presence of the oligonucleotide, and polysomes become disaggregated. In the presence of high concentrations of emetine, an inhibitor of chain elongation, reticulocyte polysomes containing an average of 4-6 ribosomes per mRNA are partially degraded to structures containing 1-4 ribosomes after incubation with the oligonucleotide. The level of association of exogenous 35S-Met-tRNAf with initiation complexes is not decreased, and under some conditions is even increased, by the oligonucleotide. When RNA is extracted from control and inhibited reticulocyte lysates and assayed for active mRNA content by retranslation in a fresh mRNA-dependent system, the results show extensive loss of template activity in the material obtained from the incubations containing pppA2′ p5′ A2′ p5′ A. The data are consistent with a mechanism in which this inhibitor activates a nuclease which prevents mRNA from being utilized for protein synthesis. This mechanism is contrasted with that of the heme-controlled repressor, another potent inhibitor of translation, which causes extensive inhibition of Met-tRNAf binding to initiation complexes, has no effect on polysome size in the presence of emetine and does not inactivate mRNA.