All types of double-stranded RNA (DSRNA) tested inhibit protein synthesis in rabbit reticulocyte lysates. The inhibition is characterized by its strongly biphasic kinetics, and can be enhanced by preincubation of the lysate with dsRNA in the absence of protein synthesis. Only properly and extensively matched dsRNA (greater than about 50 base pairs) has this property; no form of DNA, single-stranded RNA or even RNA-DNA hybrids act as inhibitors in this way. The cause of the inhibition appears to be a failure of initiator tRNA to associate with native ribosomal subunits in the initiation process (Darnbrough, C., Hunt, T., and Jackson, R. J. (1973) Biochem. Biophys. Res. Commun. 48, 1556-1564). We have shown that this block is not accompanied by stable association of dsRNA with the ribosomes. There are several reasons to believe that the mechanism of action of dsRNA may be complex with the possible involvement of at least one catalytic step. First, the lysate is inhibited by levels of dsRNA at which ribosomes are present in 100-fold excess over base pairs of dsRNA present. Second, high concentrations of dsRNA (greater than 10 mug per ml) are not inhibitory, but can in some, but not all experiments, reverse the inhibition caused by lower levels of dsRNA. Third, a lysate which has been inhibited by dsRNA, when mixed with a fresh lysate will inhibit synthesis in the mixture much more severely than would be expected from the concentration of dsRNA now present. These results indicate that low levels of dsRNA promote the formation of an inhibitor which may exist in two forms: one that is reversible by high levels of dsRNA and one that is irreversible.