An efficient method was devised to isolate temperature sensitive mutants of E. coli defective in tRNA biosynthesis. Mutants were selected for their inability to express suppressor activity after su3(+)-transducing phage infection. In virtually all the mutants tested, temperature sensitive synthesis of tRNA(Tyr) was demonstrated. Electrophoretic fractionation of (32)P labeled RNA synthesized at high temperature showed in some mutants changes in mobility of the main tRNA band and the appearance of slow migrating new species of RNA. Temperature sensitive function of mutant cells was also evident in tRNA synthes: directed by virulent phage T4 and BF23. We conclude that although the mutants show individual differences, many are temperature sensitive in tRNA maturation functions. In spite of much information on the structure and function of transfer RNA (tRNA), our knowledge concerning the biosynthesis of tRNA is relatively poor. It is generally assumed that complete tRNA molecules are made via a series of processing steps from the original transcription products of tRNA genes which are presumably unmodified and longer than mature tRNA molecules. In the case of tyrosine suppressor tRNA of su3(+), an unmodified precursor RNA carrying additional residues at the 3' and 5' ends has been isolated (1,2), and an endonuclease cleaving at the 5' side of this precursor has been identified in E. coli (3). In the case of T4 encoded tRNA, a large precursor molecule for several tRNA's has been reported (4). Some enzymes that catalyze the modifications have also been described (5). However, the over-all picture and the precise mechanisms of tRNA maturation are as yet largely unkown. For study of tRNA biosynthesis in E. coli, a genetic approach may prove useful, as has been the case in other biosynthetic pathways. In order to obtain mutants blocked in any of the intermediary steps of tRNA synthesis, we have developed an efficient selection system that enriches these mutants. Since any mutational block in tRNA biosynthesis might well be lethal, we looked for conditional lethal mutants in which the defect in tRNA synthesis occurs only at high temperature. In this selection system, the su3 gene carried by a temperate phage was newly introduced into cells(su(-)) and those cells incapable of synthesizing su3(+) tRNA at high temperature were selected. Such mutants were easily enriched by using conditions in which cells expressing suppressor activity were killed by two virulent phages. In this communication, we report the method for isolation of mutants and some characterization of tRNA synthesis in these mutants. Recently, Schedl and Primakoff (6) have independently isolated thermosensitive mutants of E. coli defective in tRNA synthesis which may or may not be different types from ours.