The primary sequence was established for two lysine tRNA isoacceptors which differ in abundance during development in Bacillus subtilis. Both tRNAs shared the same primary sequence but differed in the degree of post-transcriptional modification in the anticodon loop. The earlier eluting species, tRNA lys 1, had an unmodified C in position 32 and a mixture of N-[9-beta-ribofuranosyl) purin-6-ylcarbamoyl]-L-threonine, t6A, and N-[(9-beta-D-ribofuranosyl-2-methylthio-purin-6-yl)carbamoyl]threonine, ms2t6A, in position 37. The later eluting species, tRNA Lys 3, which is the more efficient in protein synthesis, had a modified C in position 32 and only ms2t6A in position 37. The possibility exists that modification to make a more efficient tRNA species may be part of a functional interaction between the translational and transcriptional changes that are part of the differentiation process in B. subtilis.