Various fluorescence experiments and computer simulations were utilized to gain further understanding of thromboxane A(2) synthase (TXAS), which catalyzes an isomerization of prostaglandins H(2) to give rise to thromboxane A(2) along with a fragmentation reaction to 12-L-hydroxy-5,8,10-heptadecatrienoic acid and malondialdehyde. In this study, 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS) was utilized as a probe to assess the spatial relationship and binding dynamics of ligand-TXAS interactions by steady-state and time-resolved fluorescence spectroscopy. The proximity between TNS and each of the five tryptophan (Trp) residues in TXAS was examined through the fluorescence quenching of Trp by TNS via an energy transfer process. The fluorescence quenching of Trp by TNS was abolished in the W65F mutant, indicating that Trp65 is the major contributor to account for energy transfer with TNS. Furthermore, both competitive binding experiments and the computer-simulated TXAS structure with clotrimazole as a heme ligand strongly suggest that TXAS has a large active site that can simultaneously accommodate TNS and clotrimazole without mutual interaction between TNS and heme. Displacement of TNS by Nile Red, a fluorescence dye sensitive to environmental polarity, indicates that the TNS binding site in TXAS is likely to be hydrophobic. The Phe cluster packing near the binding site of TNS may be involved in facilitating the binding of multiple ligands to the large active site of TXAS.