Ammonium 4-chloro-7-sulfobenzofurazan (Sbf-Cl) is a water-soluble fluorescent reagent which is highly specific for thiol groups. We describe here a simple and sensitive fluorescence assay which is highly specific for subunit 3 of the rat glutathione S-transferases. Specific activities of isoenzymes 3-3 and 3-4 were an order of magnitude greater than those of isoenzymes 1-1, 1-2, 2-2, and 4-4, with glutathione and Sbf-Cl concentrations of 1 and 2 mM, respectively. The catalytic specificity constant, kcat/Km was in the range of 104-106 M-1 s-1, indicating that Sbf-Cl is a very good substrate for rat hepatic glutathione S-transferases. The specificity constants measured for the heterodimers 1-2 and 3-4 were greater than the values predicted when dimeric independence is assumed. This suggests that binding of Sbf-Cl to the glutathione S-transferases may result in steric alterations and subsequent elevation in enzymatic activity. Sbf-Cl was shown to be at least 10-fold more sensitive for the detection of rat GST isoenzyme 3-3 than DCNB. Consequently, Sbf-Cl will have an important future role in the investigation of the active site topology of glutathione S-transferases, in addition to its obvious potential as a substrate for the detection and quantitation of rat glutathione S-transferase subunit 3.