3-Hydroxyflavones are characterized by an excited-state proton transfer reaction between two tautomeric excited states, which results in two emission bands well separated on the wavelength scale. Due to the high sensitivity of the relative intensities of the two emission bands to solvent polarity, hydrogen bonding, and the local electrical field, these dyes found numerous applications in biomembrane studies. In the present work, we evidenced a new band strongly shifted to the red in both absorption and excitation spectra for 4'-(dimethylamino)-3-hydroxyflavone (probe F) in the presence of anionic phosphatidylglycerol vesicles. Excitation of this new band provided a single-band emission spectrum with a maximum around 530 nm that differs from the two-band profile obtained at lower excitation wavelengths. Furthermore, an additional long-lived lifetime (3.8 ns) was observed in the time-resolved decay only when the probe was excited in the wavelength range of the new excitation band. These spectroscopic features were observed at neutral pH in HEPES but not phosphate-citrate-borate buffer. From their pH dependence, these features could be unambiguously attributed to the anionic form of probe F bound at the interface of the lipid bilayer. This anion may form even at neutral pH due to the HEPES molecules that bind at the bilayer interface to the negatively charged polar heads of phosphatidylglycerol and deprotonate probe F. Consequently, HEPES can significantly modify the acid-base properties of the negatively charged lipid membrane and thus affect the properties of phenolic compounds and other weak acids bound to lipid vesicles or cell membranes. It follows that HEPES buffer should be used with care in biomembrane studies.