The fate of host tRNAs during T4 bacteriophage infection was investigated with Escherichia coli CTr5x, the only known host strain that is restrictive to RNA ligase and polynucleotide kinase mutants. Three CTr5x tRNA species were cleaved during infection. One was leucine tRNA1, which was cleaved in the extra arm, as reported elsewhere for E. coli B infected with bacteriophage T2 or T4. The other two were specific to E. coli CTr5x and were not cleaved in various other hosts. One of the cleaved CTr5x-specific tRNAs had an anticodon sequence of the E. coli B "major" isoleucine tRNA but otherwise little sequence homology. Both CTr5x-specific tRNAs were cleaved by a distinct T4-induced endonuclease, other than that of leucine tRNA1, because the CTr5x-specific cleavages (i) were induced later in infection, (ii) persisted with a T4 mutant deficient in leucine tRNA1 endonuclease, and (iii) occurred in the anticodon loop. The specific manifestation of the anticodon-directed endonuclease activity in T4-infected E. coli CTr5x suggests roles for RNA ligase and polynucleotide kinase in processing of host tRNA species.