Abstract T-even phage-specific modification of leucyl-tRNA of Escherichia coli has been established. Leucyl-tRNA F is a modified tRNA which appears one to two minutes after infection and can be detected on a methylated albumin kieselguhr column elution profile as a distinct new peak in front of all leueyl-tRNA's. Leucyl-tRNA F isolated by MAK † † Abbreviations used: MAK, methylated albumin kieselguhr; Leu-tRNA, leucyl-tRNA (acylated form); tRNA Leu, tRNA specific to leucine (non-acylated form)—see J. Biol. Chem. 241, 532. column fractionation can not be deacylated enzymically, nor can leucine from Leu-tRNA F be exchanged with other components of tRNA Leu. Re-acylation of Leu-tRNA F can not be achieved following deacylation. This is true when either bulk tRNA containing Leu-tRNA F or isolated Leu-tRNA F is used. The results indicate that once tRNA F Leu is charged with leucine, it can not be recognized by the synthetase. A comparison of hyperchromicity, the sedimentation coefficient and Sephadex G100 column fractionation of isolated Leu-tRNA F and bulk tRNA shows that Leu-tRNA F is approximately half the size of normal tRNA. tRNA F Leu (before aminoacylation) is an unstable molecule and the conditions which weaken the hydrogen-bonded structure destroy its chargeability. Sucrose density-gradient centrifugation of bulk tRNA containing tRNA F Leu indicates that tRNA F Leu sediments at the same rate as bulk tRNA, whereas Leu-tRNA F (after aminoacylation) sediments much slower than bulk tRNA. All the results obtained indicate that tRNA F Leu (before aminoacylation) has an intact size. However, it is broken by aminoacylation or by treatments which weaken the hydrogen-bonded structure. It is suggested that tRNA F Leu is derived from tRNA F Leu by the introduction of a nick or nicks within the molecule as the result of T-even phage infection.