13CH2-ethyl labeled phosphatidylethanol (PEth), a rare naturally occurring anionic phospholipid, was used to probe the interleaflet packing density difference in small and large unilamellar phospholipid vesicles (SUVs and LUVs, respectively). The intrinsically tighter lipid packing in the inner leaflet of the SUVs resulted in the splitting of the CH2-ethyl 13C-resonance into two distinct components originating from PEth molecules residing in the inner and outer leaflets. The splitting of the 13C-NMR signal from the PEth headgroup appears to be unique among naturally occurring phospholipids. We present data suggesting that the splitting of the PEth signal reports on transleaflet packing density difference modulated by unequal electrostatic interactions and structured water on the inner and outer surfaces of the SUV. The PEth resonance splitting was insensitive to pH changes over the range 5.3-8.6 and cannot be accounted for by differences in the pKa of PEth in the inner and outer monolayers of the SUV. In 13C-NMR spectra of LUVs, where packing constraints in both monolayers are approximately similar, only a single, narrow symmetrical CH2-ethyl signal was observed, which was shifted downfield at higher PEth concentrations. The carbonyl and C3-glycerol backbone PEth resonances were shifted upfield compared to those of phosphatidylcholine or phosphatidylglycerol, suggesting a more tightly packed/hydrophobic environment for these segments of the PEth molecule in the membrane. We conclude that the unique splitting of the PEth 13C-resonance reported here can be used to characterize the lipid packing conditions in various membranes and to monitor the transbilayer distribution/movement of PEth.