An in vitro system to assay translational readthrough of the UAG termination codon at the murine leukemia virus (MuLV) gag-pol junction was developed by using rabbit reticulocyte lysates programmed by SP6-generated Moloney MuLV gag-pol mRNA. Under conditions in which the suppressor activity of the lysate was dependent on addition of tRNA, it could be shown that readthrough synthesis was stimulated to approximately the same extent by equivalent amounts of tRNA from MuLV-infected and uninfected NIH 3T3 cells. Analysis of glutamine tRNA, which mediates suppression in vivo, showed that the level of glutamine acceptor activity and the chromatographic profile of glutamine isoacceptors were unchanged following virus infection. On the basis of these results, we conclude that the suppressor tRNA occurs normally within the tRNA population of uninfected cells and need not be induced in response to virus infection.