E. coli unfractionated tRNA and tRNA phe both contain a single strong ethidium binding site. Singlet-singlet energy transfer has been used to measure the distance between this site and dansyl hydrazine covalently attached to the 3' end of the tRNAs. The distance obtained is between 33 and 40 A for both samples. This is completely consistent with results from earlier NMR studies which placed the single, strong ethidium binding site of yeast tRNAphe between base pairs 6 and 7 on the aminoacyl stem. From the known tertiary structure of tRNAphe it is possible to rationalize the unusual affinity of this site and its likely existence in all tRNAs.