Fourteen different DNA fragments containing Escherichia coli tRNA genes have been cloned using the vector pBR322. We report the methods of cloning, the identification of specific tRNA genes, and the presence or absence of rRNA genes on these cloned DNA fragments. In particular, one chimeric plasmid contains a 17.0 kilobase pair EcoRI fragment bearing tRNA(Leu 1) sequences. Using nucleotide sequence analysis we have identified a cluster of three tandem tRNA(Leu 1) genes separated by intergenic spacers of 27 and 34 base pairs, respectively. The nucleotide sequence upstream of the first gene contains a transcription promoter site. A G-C rich sequence (5'-CGCCTCC-3') found between the Pribnow box and initiation site is very similar to the corresponding sequence found in other genes which are under stringent control.