The symmetrical dimer structure of tryptophanyl-tRNA synthetase is similar to that of tyrosyl-tRNA synthetase whose binding behavior and structural details have been elucidated in detail. The structure of both subunits after forming the intermediate tryptophanyl-AMP has important implications for the binding of the cognate tRNATrp. Single tryptophan mutants of Bacillus stearothermophilus tryptophanyl-tRNA synthetase have been constructed and expressed and used to probe structural changes in different domains of the enzyme in both subunits. Substrate titrations using the Trp analogues 4-fluorotryptophan and 7-azatryptophan in the presence of ATP to form the corresponding aminoacyl-adenylate reveal significant structural changes occurring throughout the active subunit in regions not confined to the active site. Changes in environment around the specific Trp residues were monitored using UV absorbance and steady-state fluorescence measurements. When titrated with 4-fluorotryptophan, both Trp 91 and Trp 290 fluorescence is quenched (49 and 22%, respectively) when one subunit has formed Trp-AMP. The fluorescence of Trp 48 is enhanced 19%. No further change in signal was observed after a 1:1 dimer/l-4FW-AMP complex ratio had been established. Using an anion-exchange filter binding assay with radiolabeled l-Trp as a substrate, binding to only one subunit was observed under nonsaturating conditions. This agrees with the results of the assay using 7-azatryptohan as a substrate. The observed changes extend to the unfilled subunit where a similar structure is believed to form after one subunit has formed tryptophan-AMP. Movement in the regions of the enzyme containing Trp 290 and Trp 91 suggests a mechanism for cross-subunit communication involving the helical backbone and dimer interface containing these two residues.