Steady-state and time-resolved techniques were employed to study the excited-state proton transfer (ESPT) from d-luciferin, the natural substrate of the firefly luciferase, to the mild acetate base in aqueous solutions. We found that in 1 M aqueous solutions of acetate or higher, a proton transfer (PT) process to the acetate takes place within 30 ps in both H2O and D2O solutions. The time-resolved emission signal is composed of three components. We found that the short-time component decay time is 300 and 600 fs in H2O and D2O, respectively. This component is attributed either to a PT process via the shortest water bridged complex available, ROH··H2O··Ac-, or to PT taking place within a contact ion pair. The second time component of 2000 and 3000 fs for H2O and D2O, respectively, is attributed to ROH* acetate complex, whose proton wire is longer by one water molecule. The decay rate of the third, long-time component is proportional to the acetate concentration. We attribute it to the diffusion-assisted reaction as well as to PT process to the solvent.