Transfer RNA (tRNA) from all organisms on this planet contains modified nucleosides, which are derivatives of the four major nucleosides. tRNA from Escherichia coli/Salmonella enterica serovar Typhimurium contains 33 different modified nucleosides, which are all, except one (Queuosine [Q]), synthesized on an oligonucleotide precursor, which by specific enzymes later matures into tRNA. The structural genes for these enzymes are found in mono- and polycistronic operons, the latter of which have a complex transcription and translation pattern. The synthesis of the tRNA-modifying enzymes is not regulated similarly, and it is not coordinated to that of their substrate, the tRNA. The synthesis of some of them (e.g., several methylated derivatives) is catalyzed by one enzyme, which is position and base specific, whereas synthesis of some has a very complex biosynthetic pathway involving several enzymes (e.g., 2-thiouridines, N<sup>6</sup>-cyclicthreonyladenosine [ct<sup>6</sup>A], and Q). Several of the modified nucleosides are essential for viability (e.g., lysidin, ct<sup>6</sup>A, 1-methylguanosine), whereas the deficiency of others induces severe growth defects. However, some have no or only a small effect on growth at laboratory conditions. Modified nucleosides that are present in the anticodon loop or stem have a fundamental influence on the efficiency of charging the tRNA, reading cognate codons, and preventing missense and frameshift errors. Those that are present in the body of the tRNA primarily have a stabilizing effect on the tRNA. Thus, the ubiquitous presence of these modified nucleosides plays a pivotal role in the function of the tRNA by their influence on the stability and activity of the tRNA.