Abstract— The odd nucleoside 4‐thiouridine, which is present in position 8 of 70% of E. coli tRNAs, possesses unusual spectroscopic properties which make it suitable for intramolecular energy transfer studies. Both its luminescence excitation spectrum and the action spectrum (230–380 nm) for the 8–13 link formation have been established in native E. coli tRNA at room temperature. The spectra are identical and present a new unexpected peak around 260 nm. At this wavelength, they are amplified by a factor of nine as compared with the absorption and excitation spectra of the free nucleoside in aqueous solution.The origin of this new peak is discussed and it is concluded that energy transfer does occur from the common nucleosides to the 4‐thiouridine residue. Using the values of the nucleosides to 4‐thiouridine distances inferred from the sets of atomic coordinates obtained on yeast tRNAphe crystals, a satisfactory account of our finding can be obtained assuming singlet‐singlet energy transfer. The efficiency of the mechanism is probably favoured by a good overlap between the emission spectra of the common nucleosides and the absorption spectrum of 4‐thiouridine.