This distance between a base next to the anticodon of tRNA and the 3' CpCpA terminus of the molecule has been estimated by singlet-singlet energy transfer experiments. The energy donor was the Y base of unknown structure found in yeast tRNA(phe). Three different energy acceptors were used: acriflavine, proflavinyl acetic acid hydrazide, and 9-hydrazino acridine. These were attached to the periodate-oxidized 3' end of the tRNA. R(0)'s between 24 and 30 A were calculated for the three chromophore couples by assuming that the relative orientation of donor and acceptor is random. This assumption is supported by the consistency of the experimental results with all three acceptors and by studies of the fluorescence depolarization of Y. The energy transfer observed both by quenching of Y and enhanced activation of the acceptors is quite small, indicating that the anticodon is more than 40 A away from the amino acid accepting terminus. This places severe restrictions on the type of tertiary structure possible for tRNA.