Inhibition of protein synthesis in rabbit reticulocyte lysates by double-stranded RNA is caused by the inactivation of IF-3, an initiation factor required for the recycling of ribosomes and for their binding to messenger RNA. The evidence for this is that (i) the inhibition can be overcome by addition of exogenous If-3; (ii) double-stranded RNA inactivates stoichiometric amounts of IF-3; (iii) double-stranded RNA forms a complex with IF-3; and (iv) double-stranded DNA, which lacks inhibitory activity, also binds to IF-3, but with a much lower affinity than double-stranded RNA. It is concluded that double-stranded RNA inhibits cellular protein synthesis by tightly complexing with IF-3. It is suggested that IF-3 normally recognizes a double-stranded region in messenger RNA.