To elucidate subtle functions of transfer ribonucleic acid (tRNA) modifications in protein synthesis, pairs of tRNA's that differ in modifications at specific positions were prepared from Bacillus subtilis. The tRNA's differ in modifications in the anticodon loop, the extra arm, and the TWC loop. The functional properties of these species were compared in aminoacylation, as well as in initiation and peptide bond formation, at programmed ribosomes. These experiments demonstrated the following. (i) In tRNAMet the methylation of guanosine 46 in the extra arm to 7-methylguanosine by the 7-methylguanosine-forming enzyme from Escherichia coli changes the aminoacylation kinetics for the B. subtilis methionyl-tRNA synthetase. In repeated experiments the Vmax value is decreased by onehalf. (ii) tRNAfmet species with ribothymidine at position 54 (rT54) or uridine at position 54 (U54) were obtained from untreated or trimethoprim-treated B. subtilis. The formylated fMet-tRNAPet species with U54 and rT54, respectively, function equally well in an in vitro initiation system containing AUG, initiation factors, and 70s ribosomes. The unformylated MetAtRNAmet species, however, differ from each other: "Met-tRNAm't rT" is inactive, whereas the U54 counterupart effectively forms the initiation complex. (iii) Two isoacceptors,tRNAlhe and tRNA he, were obtained from B. subtilis. tRNAIhe accumulates only under special growth conditions and is an incompletely modified precursor oftRNA2he: in the first position of the anticodon, guanosine replaces Gm, and next to the 3' end of the anticodon (isopentenyl)adenosine replaces 2-thiomethyl-N6-(isopentenyl)adenosine. Both tRNA's behave identically in aminoacylation kinetics. In the factor-dependent AUGU3-directed fornation of fMet-Phe, the undermodified tRNAphe is always less efficient at Mg2+ concentrations between 5 and 15 mM than its mature counterpart.