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 V-3 antisymmetric stretching mode of N2O 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 v(3) band (similar to 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.