Sequences encoding eight tRNAs and two stable RNAs of bacteriophage T4 are grouped together on the T4 genome in two clusters, separated by approximately 500 base pairs. The DNA sequence of part of this region was determined. Within each cluster coding sequences are separated by only one or a few base pairs. These findings imply that the RNAs may be processed from a single multimeric transcript, with initial endonucleolytic cleavages generating the previously characterized monomeric and dimeric precursors.The tRNAs encoded by bacteriophage T4 have provided a useful system for the study of tRNA biosynthesis. Bacteriophage T4 encodes eight tRNAs and two low molecular weight stable RNAs of unknown function. The nucleotide sequences of small precursor RNAs for most of these have been determined (ref. 1 and references cited therein; unpublished data), and the synthetic steps leading from these intermediates to the mature molecules are known (2). Less is known about the initial steps responsible for formation of the small precursor RNAs. We know that the small precursor RNAs are not primary transcripts, and evidence from in vitro transcription studies of the T4 tRNA coding sequences (3), which are grouped together in a small region of the T4 genome ( Fig. 1) (4), indicates that these sequences are cotranscribed. However, attempts to isolate this large transcript from infected cells have thus far been unsuccessful. In the present study, restriction endonuclease fragments containing the coding sequences of seven of the stable RNAs were inserted into a plasmid vector and cloned to allow determination of their sequences. The sequence information thus obtained allows us to predict the entire biosynthetic pathway leading from the coding sequences to the mature RNAs. (Fig. 1) in the plasmid vector pBR322. Cloning procedures are given elsewhere (6). All cloning procedures followed the National Institutes of Health guidelines.An EcoRI fragment carrying the sequences of tRNAArg, band D RNA, and band C RNA was inserted in both orientations into the unique EcoRI site of pBR322. Cleavage of these two recombinant plasmids at the unique HindIII site of pBR322, followed by exonuclease III digestion, generated two molecules in which opposite strands of the cloned DNA fragments were * Present address: