3,3'5,5'-Tetramethylbenzidine (TMB) has been shown to be a safe-sensitive substrate for the assay of horseradish peroxidase (HRP). Initially, in the presence of HRP and hydrogen peroxide, a one-electron oxidation product is formed. This compound, a cation free radical, is blue in color with an adsorption maximum at 653 nm. Further reaction with HRP/H?O? or acidification of the radical with acid yields the diimine terminal oxidation product adsorbing light at 450 nm. The extinction coefficient of the radical (E653 nm = 3.9 x 104 mo?11 cm?1) and diimine (E450 nm = 5.9 x 104 mol?1 cm?1) provide a remarkably sensitive system for the assay of HRP and HRP labeled probes. TMBUS, available from Biovision Inc., is a single component reagent stable a