Photochemical mapping of nucleophiles in close proximity to the active site Cys149 of rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was demonstrated based on the nucleophilic aromatic photosubstitution reaction using two regioisomers of alkoxy-fluoro-nitro-substituted benzenes. Two photophores were covalently attached to the active site SH group of GAPDH and the protein was subjected to photolysis then to the cyanogen bromide cleavage reaction. The advantage of this method is the capability to chase labeled products by monitoring absorption at 380 nm because of the chromogenic property of photophore. HPLC separation identified a large labeled peptide fragment that was further digested by V8 protease for Edman sequence analysis. From the recent X-ray crystallography of rabbit GAPDH, Tyr311, His176, Ser238 and Lys183 are closely located to catalytic Cys149. Among these nucleophiles, Tyr311 was preferentially labeled with 2-fluoro-4-nitrophenoxy photophore and no label was identified with the isomeric 4-fluoro-2-nitrophenoxy photophore. The result clearly reflects the distance between Cys149 and nucleophiles to distinguish the nearest Tyr311. As photophores show great reactivity even with water under neutral conditions, the distance between nucleophiles and photophores is important for photo-induced nucleophilic aromatic substitution. The method will provide a useful technique to survey nucleophiles within the catalytic domain.