Ribothymidine (m5u) in tRNAs of M. lysodeikticus is not derived from methionine. The results indicate that as in tRNAs of B. subtilis a tetrahydrofolate derivative is involved in the formation of m5U, whereas methionine serves as precursor in the biosynthesis of m7G, m1A and m6A. Ribothymidine also occurs in 23S rRNA of B. subtilis and M. lysodeikticus. Approximately 2-3 moles of m5U residues were found per mole of 23S rRNA. In contrast to m5U residues present in tRNAs of B. subtilis and M. lysodeikticus, ribothymidine in 23S rRNA of these organisms and of E. coli is synthesized via S-adenosylmethionine. m6A and m1G, present in E. coli rRNAs, were not detected in rRNAs of (methyl-14C) methionine labeled B. subtilis and M. lysodeikticus.