Abstract Inactive, frozen and thawed cytoplasmic extracts of 3T3 and SV-101 (3T3 transformed by SV-40 virus) cells contain an inhibitor which blocks the poly(U)-directed incorporation of [ 14C]phenylalanine into polypeptides, catalyzed by active extracts of these cells. This inhibition is not reversed by adding increased amounts of poly(U). Furthermore, little or no inhibitory activity is observed when poly(U) translation is assayed using precharged [ 14C]Phe-tRNA. These results suggest that the observed inhibition is not due to the degradation of poly(U) by a nuclease. The inhibitor appears to act primarily at the level of tRNA charging since the synthesis of both Phe-tRNA and Lys-tRNA is impaired in its presence. Evidence is presented which indicates that the inhibitory activity is not due to a high molecular weight protein or nucleic acid. However, the inhibitor appears to be adsorbed to a macromolecule. The inhibitory activity is completely destroyed by ashing.