Experimental time-resolved spectral and photon counting kinetic results confirm formation of an isoalloxazinicexcited state via excited-state double proton transfer (ESDPT) catalyzed by a carboxylic acid molecule thatforms a hydrogen-bond complex with the parent alloxazine molecule. This isoalloxazinic tautomer manifestsitself as a distinct long-lived emissive species formed only in such alloxazine derivatives that were notsubstituted at the N(1) nitrogen atom, being a product of the excited-state reaction occurring from the alloxazinicexcited state. Theoretical calculations support the idea that the ESDPT occurs by the concerted mechanism.The calculated activation barrier in the excited state is much lower than the same barrier in the ground stateand even disappears for the HOMO-1 to LUMO excitation, which explains the fact that the reaction takesplace in the excited-state only. The reaction rate estimated from the emission kinetics is ca. 1.4 × 108 dm3mol-1 s-1 in ethanolic solutions of lumichrome with added acetic acid.