The effect of (p)ppGpp on the accuracy of translation was studied in vitro using a poly(U)-programmed poly(Phe)-synthesizing system operating at incorporation rates and missense error frequencies close to the values obtained in vivo. Simulation of a relaxed phenotype in vitro was accomplished by limitation of the cognate aminoacyl-tRNA synthetase, while the noncognate aminoacyl-tRNA synthetase was included at saturating concentrations. This protocol yielded a Phe-tRNA-starved steady state system displaying the expected decrease in Phe polymerization rates accompanied by a drastic increase in relative Leu misincorporation errors. The use of purified enzymes permitted us to assay for the effects of the individual nucleotides ppGpp and ppGpp as well as their potential targets, the elongation factors Tu and G, upon the missense error rates. Our results support the conclusion that ppGpp reduces misincorporation in a starved in vitro system by preferentially inhibiting EF-Tu. The details of the proposed mechanism and their relevance to an in vivo situation are discussed.