Histamine reacts with o-phthalaldehyde (OPT) to form colored as well as highly fluorescent derivatives. After large-scale synthesis, the chromophores were eliminated and the pale yellowish fluorophore was isolated by a series of chromatographic steps, followed by semipreparative high-performance liquid chromatography. Structural analysis of the fluorophore was performed using NMR and mass spectrometry. The isolated fluorophore was found to be a condensation product of 2 molecules of OPT and 1 molecule of histamine, arranged to form a complex dihydrophenantroline derivative with a quaternary amine group. The molecular weight of its chloride salt was 379.5. It was shown that the fluorophore exists in aqueous milieu as an equilibrium between three isomers.