The fluorescent probe 1,5-I-AEDANS was covalently attached to bovine tyrosyl-tRNA synthetase outside of enzyme active site in a nearly stoichiometric amount (2 probe molecules per enzyme dimer). Singlet-singlet resonance energy transfer has been used for the measurement of the apparent distance between tryptophan residues of enzyme and covalently attached 1,5-I-AEDANS. This distance was estimated as 27.4 A in the assumption of the random orientation of the donor and acceptor fluorophores. Tyrosyl adenylate formation catalyzed by bovine tyrosyl-tRNA synthetase resulted in the highly specific enhancement of 1,5-I-AEDANS fluorescence and concomitant decrease of the apparent distance between the probe and tryptophanyls to 22.3-25.7 A. These results are consistent with the conformational change of tyrosyl-tRNA synthetase during tyrosyl adenylate formation which propagates to distant from active site regions of enzyme structure.