In aqueous alkaline solutions, the potential-resolved electrochemiluminescent (ECL) behavior of calcein at a glassy carbon electrode was investigated. A sharp ECL light emission peak appeared near 0.8V (vs. Ag/AgCl) under cyclic voltammogram (CV) conditions. In order to obtain a better understanding of the light emission, we investigated the effects of supporting electrolytes, the pH value, the presence of O-2 or N-2, and the concentration of calcein. The oxidative form of calcein reacted with hydroxyl radicals and oxygen, producing singlet excited oxygen that was identified as the emitter using the ECL, UV-vis absorption and fluorescence spectra. A possible mechanism for the behavior of calcein in this system was also proposed. (c) 2008 Elsevier Ltd. All rights reserved.