The interactions of neuropeptides and membranes play an important role in peptide hormone function. Our current understanding of peptide-membrane interactions remains limited due to the paucity of experimental techniques capable of probing such interactions. In this work, we study the nature of opioid peptide-membrane interactions using ultrafast two-dimensional infrared (2D IR) spectroscopy. The high temporal resolution of 2D IR is particularly suited for studying highly flexible opioid peptides. We investigate the location of the tyrosine (Tyr) side chain of leucine-enkephalin (Lenk) in lipid bilayer membranes by measuring spectral diffusion of the phenolic ring vibrational mode in three different systems: Lenk in lipid bilayer membranes (bicelles), Lenk in deuterated water, and p-cresol in deuterated water. Frequency-frequency correlation functions obtained from waiting-time-dependent 2D IR spectra reveal an ultrafast decaying component with an approximately 1 ps time constant that is common for all three systems. On the basis of density functional theory calculations and molecular dynamics simulations, this spectral diffusion component is attributed to hydrogen-bond dynamics of the phenolic hydroxyl group interacting with bulk water. Unlike p-cresol in water, both Lenk systems exhibit static spectral inhomogeneity, which can be attributed to conformational distributions of Lenk that do not interconvert within 4 ps. Our results suggest that the Tyr side chain of Lenk in bicelles is located at the water-abundant region at the membrane-water interface and not embedded into the hydrophobic core.