Steady-state absorption and emission as well as time-resolved emission spectroscopies were employed to study the photophysics and photochemistry of D-luciferin, the firefly active bioluminescent compound. In aqueous solution the electronically excited-state protonated D-luciferin compound undergoes an efficient process of proton transfer to the solvent, with a rate constant k(PT) = 3.0 x 10(10) s(-1). We found a kinetic isotope effect of about 2.5 for this process. The deprotonated form of D-luciferin in the excited state recombines irreversibly with the geminate proton. Hence, the fluorescence decay of the deprotonated form is nonexponential, and the fluorescence quantum yield is low.