Two genes, tufA and tufB, located at 73 and 88 minutes of the Escherichia coli linkage map, code for the polypeptide chain elongation factor EF-Tu. tufB is transcribed with four upstream tRNA genes, thrU, tyrU, glyT and thrT, into a cotranscript of approximately 1800 nucleotides. Here we show that in vivo processing yields a 1320 nucleotide transcript of tufB. S1 nuclease fine mapping reveals that the processing site is located in the intergenic region at about 72 to 74 nucleotides upstream from the initiation codon of the tufB cistron. A deletion in the cloned tRNA-tufB operon, encompassing the 3' half of thrU, the complete tyrU, glyT, thrT genes and ten nucleotides of the intergenic region, causes a threefold increase of the rate of plasmid tufB transcription, a fourfold increase of plasmid-borne tufB RNA and a twofold increase of plasmid-borne EF-TuB. We conclude that the deletion has eliminated a transcription termination site probably located after the thrT gene. Termination at this site uncouples tRNA synthesis from tufB transcription.