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 contains 31 different modified nucleosides, which are all, except for one (Queuosine[Q]), synthesized on an oligonucleotide precursor, which through specific enzymes later matures into tRNA. The corresponding structural genes for these enzymes are found in mono- and polycistronic operons, the latter of which have a complex transcription and translation pattern. The syntheses of some of them (e.g.,several methylated derivatives) are catalyzed by one enzyme, which is position and base specific, but synthesis of some have a very complex biosynthetic pathway involving several enzymes (e.g., 2-thiouridines, N 6 -threonyladenosine [t6A],and Q). Several of the modified nucleosides are essential for viability (e.g.,lysidin, t 6 A, 1-methylguanosine), whereas deficiency in 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, which are present in the body of the tRNA, have a primarily stabilizing effect on the tRNA. Thus, the ubiquitouspresence 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.