Steady-state and time-resolved emission techniques were used to study the protolytic processes in the excited state of dehydroluciferin, a nonbioluminescent product of the firefly enzyme luciferase. We found that the ESPT rate coefficient is only 1.1 × - 1010 s-1, whereas those of d-luciferin and oxyluciferin are 3.7 × - 1010 and 2.1 × - 1010 s-1, respectively. We measured the ESPT rate in water-methanol mixtures, and we found that the rate decreases nonlinearly as the methanol content in the mixture increases. The deprotonated form of dehydroluciferin has a bimodal decay with short- and long-time decay components, as was previously found for both d-luciferin and oxyluciferin. In weakly acidic aqueous solutions, the deprotonated forms emission is efficiently quenched. We attribute this observation to the ground-state protonation of the thiazole nitrogen, whose pKa value is ∼3.