Escherichia coli strains mutated in the relA gene lack the ability to produce ppGpp during amino acid starvation. One consequence of this deficiency is a tenfold increase in misincorporation at starved codons compared to the wild-type. Previous work had shown that the charging levels of tRNAs were the same in Rel⁺ and Rel^- strains and reduced, at most, two- to fivefold in both strains during starvation. The present reinvestigation of the charging levels of tRNA₂^Arg, tRNA₁^Thr, tRNA₁^Leu and tRNA^His during starvation of isogenic Rel⁺ and Rel^- strains showed that starvation reduced charging levels tenfold to 40-fold. This reduction corresponds much better with the decreased rate of protein synthesis during starvation than that reported earlier. The determination of the charging levels of tRNA₂^Arg and tRNA₁^Thr during starvation were accurate enough to demonstrate that charging levels were at least fivefold lower in the Rel^- strain compared to the Rel⁺ strain. Together with other data from the literature, these new data suggest a simple model in which mis-incorporation increases as the substrate availability decreases and that ppGpp has no direct effect on enhancing translational accuracy at the ribosome.