An E. coli mutant that lacks tRNA-guanine transglycosylase was isolated by random screening from a collection of Escherichia coli mutants obtained with N-methyl-N'-nitro-N-nitrosoguanidine. The defective gene, named tgt, was mapped at about 9 min on the E. coli chromosome, and the gene order was shown to be phoB-tgt-tsx. tgt was transferred to an E. coli strain with a defined genetic background by P1 transduction to investigate its function. The mutant thus obtained lacked queuosine (2-amino-5-[3S, 4R, 5S)-4,5-dihydroxycyclopent-1-en-3-ylaminomethyl]-7-(beta-D-ribofuranosyl)-pyrro lo-[2,3-D]-pyrimidin-4-one) in tRNA, indicating that the enzyme is actually involved in the biosynthesis of queuosine in tRNA. No clear biological defect was observed in the mutant, and, in fact, it grew slightly faster than the control isogenic strain. tRNATyr lacking queuosine, isolated from the mutant, showed no significant biological difference from normal queuosine-containing tRNA in amino acid acceptor activity or amino acid transfer in a cell-free protein synthesizing system directed by synthetic polynucleotide. The only phenotypic change observed in the mutant thus far is marked reduction of viability when the cells are kept under unsuitable conditions for growth, suggesting that the presence of queuosine in tRNA is important to E. coli for survival in the natural environment.