We studied the mechanisms of excited-state intramolecular proton transfer (ESIPT) and ground-state back proton transfer (BPT) in 3-hydroxyflavone (3HF) at cryogenic temperatures. The focus was on substituents that change the distribution of electronic density on the chromophore and their influence on these reaction rates. Shpol'skii spectroscopy was applied for comparative studies of three compounds:  3HF, 3-hydroxy-4‘-methoxyflavone (3HF-4‘OMe), and 2-furyl-3-hydroxychromone (3HC−F). By comparing the spectral bandwidths with those of deuterated analogues, we could distinguish the lifetime broadening components in the high-resolution excitation and emission spectra, from which the time constants of the ESIPT and BPT reactions were calculated. The time constants for the ESIPT reaction were 0.093 ps for 3HF, 0.21 ps for 3HF-4‘OMe, and slower than 0.6 ps for 3HC−F. For the same compounds, the BPT rates were 0.21, 0.47, and >2 ps, respectively. No change in bandwidth was observed over the temperature range 4−20 K, in agreement with a tunneling mechanism. Estimates for the barrier heights and proton-transfer distances are given. In addition, a systematic change in O−H bond strengths between ground and excited states was calculated from the isotope effect, observed as the shifts of the 0−0 bands in the excitation and emission spectra upon deuteration. The substantial effect of electron donating substituents on the rates of ESIPT and BPT reactions is in agreement with these changes.