The Escherichia coli tRNA(Tyr) gene carried by the varphi80psu(3) + transducing phage was transcribed in vitro by DNA-dependent RNA polymerase. The enzymatically synthesized tRNA(Tyr)-like polynucleotide chains were detected by competition with purified E. coli(32)P-tRNA(Tyr) for specific hybridization sites on varphi80psu(3) + DNA. Analysis by sucrose gradient centrifugation showed the tRNA(Tyr)-like chains to possess a heterogeneous distribution with respect to sedimentation coefficients, with a broad peak around 8 S. The presence of the termination factor rho during the transcription did not significantly reduce the average size of the in vitro synthesized tRNA(Tyr)-like chains.