The structure and organization of the gene clusters coding for the two tyrosine-accepting tRNA species (tRNA1Tyr and tRNA2Tyr) on the E. coli chromosome have been determined. The mature structural sequences of the two tRNATyr genes, located on opposite sides of the E. coli chromosome, differ by only 2 bp, but sequences surrounding these portions of the genes are very different. The genes coding for tRNA1Tyr (tyrT) comprise two mature structural sequences separated by a 200 bp "intergenic spacer." It is known that in transducing phage, the region adjoining the CCA end of the second mature structural sequence comprises a 178 bp repeated sequence which contains an in vitro, rho-dependent transcriptional termination site. We find that these potentially genetically unstable repeated sequences are present in the E. coli chromosome with the same organization as that determined from transducing phage analyses. The gene that codes for tRNA2Tyr (tyrU) is present in a single copy and is tightly clustered with three other tRNA genes. One of these genes (to be called thrU) encodes a previously undescribed tRNA (to be called tRNA4Thr). The organization of this cluster on the E. coli chromosome is tRNA4Thr--8 bp--tRNA2Tyr--115 bp--tRNA2Gly--6 bp--tRNA3Thr. The importance of correlating structural analyses derived from specialized transducing phage with those determined for the chromosome itself is demonstrated by results which show that out of four independently isolated tRNATyr transducing phage, two carrying the tRNA1Tyr genes [phi80psu3+,- (Cambridge) and phi80sus2psu3+ (Kyoto)] and two carrying the tRNA2Tyr gene (lambdarifd 18 and lambdah80dglyTsu+36), only the first phage from each group has the same gene organization as that found in the E. coli chromosome.