Synthesis of T4 tRNA Gln depends on normal levels of Escherichia coli ribonuclease III. Infection of cell strains carrying a mutation in the gene for this enzyme resulted in severe depression in tRNA Gln production, as revealed by chemical and suppressor tRNA analyses. The remaining seven T4 tRNAs were synthesized in the mutant cells. The requirement of ribonuclease III for synthesis of tRNA Gln points to an essential cleavage by the enzyme of a precursor RNA containing tRNA Gln.