Ultrafast infrared spectroscopy of N2O is shown to be a sensitive probe of hydrophobic and aqueous sites in lipid bilayers. Distinct rates of VER of the ν3 antisymmetric stretching mode of N 2O can be distinguished for N2O solvated in the acyl tail, interfacial water, and bulk water regions of hydrated dioleoylphosphatidylcholine (DOPC) bilayers. The lifetime of the interfacial N2O population is hydration-dependent. This effect is attributed to changes in the density of intermolecular states resonant with the ν3 band (∼2230 cm-1) resulting from oriented interfacial water molecules near the lipid phosphate. Thus, the N2O VER rate becomes a novel and experimentally convenient tool for reporting on the structure and dynamics of interfacial water in lipids and, potentially, in other biological systems. © 2008 American Chemical Society.