Double-stranded ribonucleic acid (dsRNA) is a powerful inhibitor of initiation of protein synthesis in rabbit reticulocyte lysates. This paper shows that translation of Mengovirus or Coxsackie virus RNA in a template-dependent reticulocyte lysate is resistant to inhibition by dsRNA in conditions where translation of globin or ascites tumor cell messenger RNA (mRNA) is sensitive. The inhibition of globin mRNA translation by dsRNA is reversed completely upon addition of eukaryotic initiation factor 2 (eIF-2). During dsRNA-induced inhibition of globin mRNA translation, formation of (2'-5')-oligoisoadenylate was not detectable. The unabated translation of Mengovirus RNA in the presence of dsRNA is dependent on continued initiation: not only is translation of Mengovirus RNA as sensitive as that of globin mRNA to the specific inhibitors of initiation, pactamycin and aurin tricarboxylate, but unlike for globin mRNA the Mengovirus RNA directed incorporation of TV-formyl [35S]- methionyl-tRNAf into protein is not inhibited by dsRNA. The resistance of Mengovirus RNA translation to dsRNA is not caused by a lesser dependence on initiation factors, but by a failure of dsRNA to establish inhibition when Mengovirus RNA is used as messenger. The nature of the mRNA being translated is a critical factor in the formation of the dsRNAactivated inhibitor of translation. Mengovirus or Coxsackie virus RNA, it is shown, prevents the formation of dsRNAactivated inhibitor, while globin mRNA does not. Yet, once it is allowed to form, the inhibitor is as effective in blocking translation of Mengovirus RNA as it is in blocking translation of globin mRNA, when assayed in the presence of noninhibitory, high concentrations of dsRNA. During translation of globin mRNA in the template-dependent lysate, the presence of dsRNA stimulates the phosphorylation of the small subunit of eIF-2, as well as a 67 000-dalton polypeptide, that is characteristic of the state of translational inhibition. By contrast, when Mengovirus RNA is used as template, the dsRNA-dependent phosphorylation of either polypeptide is significantly depressed. Moreover, in a ribosomal system containing eIF-2, dsRNA, and ATP, Mengovirus RNA inhibits the phosphorylation of the small subunit of eIF-2 and of the 67 000-dalton polypeptide, apparently in a competitive manner. Globin mRNA does not possess this property. Direct RNA-binding competition studies reveal that dsRNA competes with mRNA for eIF-2, binding this factor more strongly than globin mRNA, but more weakly than Mengovirus RNA. A molecule of Mengovirus RNA exhibits a 30-40-fold greater affinity for eIF-2 than does a molecule of globin mRNA. The progressively greater affinities of globin mRNA, dsRNA, and Mengovirus RNA for eIF-2 are also reflected by the ability of these RNA species to competitively inhibit the binding of methionyl-tRNAf to eIF-2. These findings demonstrate mRNA specificity in the inhibitory action of dsRNA on eukaryotic protein synthesis. The correlation between the results of translation, inhibitor formation, phosphorylation, and binding competition experiments suggests that the affinity of a given mRNA species for eIF-2 is crucial in determining the sensitivity of its translation to dsRNA. Our results support the concept that the rate-determining event in the establishment of inhibition of translation by dsRNA involves competition between dsRNA and mRNA and that inhibitor formation, phosphorylation, and inactivation of eIF-2 depend on the outcome of this competition.