Expression of the bacteriophage lambda two-codon, AUG AUA, barI minigene (bar+) leads to the arrest of protein synthesis in cells defective in peptidyl-tRNA hydrolase (Pth). It has been hypothesized that translation of the bar+ transcript provokes premature release and accumulation of peptidyl-tRNA (p-tRNA). Inhibition of protein synthesis would then result from either starvation of sequestered tRNA or from toxicity of accumulated p-tRNA. To test this hypothesis and to investigate the cause of arrest, we used a coupled in vitro transcription-translation system primed with DNA containing bar+ and the beta-lactamase-encoding gene of the vector as a reporter. The results show that expression of bar+ minigene severely inhibits beta-lactamase polypeptide synthesis by Pth-defective extracts and partially inhibits synthesis by wild-type extracts. Fractions enriched for Pth, or a homogeneous preparation of Pth, prevented and reversed bar+-mediated inhibition. A mutant minigene, barA702, which changes the second codon AUA (Ile) to AAA (Lys), was also toxic for Pth-defective cells. Expression of barA702 inhibited in vitro polypeptide synthesis by Pth-defective extracts and, as with bar+, exogenous Pth prevented inhibition. Addition of pure tRNALys prevented inhibition by barA702 but not by bar+. Expression of bar+ and barA702 led to release and accumulation of p-tRNAIle and p-tRNALys respectively but bar+ also induced accumulation of p-tRNALys. Finally, bar+ stimulated association of methionine with ribosomes probably as fMet-tRNAfMet and the accumulation of methionine and isoleucine in solution as peptidyl-tRNA (p-tRNA). These results indicate that minigene-mediated inhibition of protein synthesis involves premature release of p-tRNA, misincorporation of amino acyl-tRNA, accumulation of p-tRNAs and possibly sequestration of tRNAs.