Fluorescence of each reaction mixture of BrCN or ferricyanide and benzylimidazolothiamine (I), imidazolothiamine (II), phenyltriazinothiamine (III) or triazinothiamine (IV) in alkaline solution was examined by fluorometer (Hitachi model 203). I showed blue fluorescence on the reaction with BrCN or ferricyanide and III or IV showed purple fluorescence by ferricyanide, but II showed no fluorescence. The fluorescence from I with BrCN was much stronger than that from I, III or IV with ferricyanide. From the reaction mixture of I and BrCN, a crystalline fluorophore was isolated and the calibration curve of its fluorescence on the basis of mole-concentration to I was shown to be nearly identical with that of I solution reacted with BrCN. As the quantitative formation of thiamine on incubation II and 4-methyl-5-(2-hydroxyethyl)-thiazole (MHT) with the cells of Saccharomyces carlsbergensis had been established, estimation of II was enabled through the calibration of the formed thiamine (thiochrome method). The method was also applied successfully for the estimation of III or IV based on the biosynthesis of thiamine from them and MHT.