Phosphatidylethanol (PtdEth) is a rare anionic phospholipid formed as a metabolite of ethanol through a transphosphatidylation reaction which is catalyzed by phospholipase D (PLD). The transbilayer distribution of dilute (0.5-2%) levels of PtdEth in small and large unilamellar phosphatidylcholine vesicles (SUVs and LUVs, respectively) was monitored by C-13 nuclear magnetic resonance spectroscopy of PtdEth specifically C-13-labeled at its headgroup methylene. Discrimination of interleaflet sites was based on shifts in the resonance frequencies of the headgroup methylene induced by differential packing in SUVs and induced by the shift reagent Pr3+ in LUVs. PtdEth is shown to undergo a rapid and reversible transbilayer redistribution (t(0.5) less than or equal to 1 h at 26 C-degrees) in response to mM concentrations of added external multivalent cations (Ca2+, Pr3+, Mn2+, and Yb3+). The pH of the vesicle suspension vastly exceeds the pK of PtdEth (ca. 1.43), based on the pH dependence of the labeled site’s chemical shift in LUVs. The rate of transbilayer transfer greatly exceeds, by 1-3 orders of magnitude, the transfer rates reported for other naturally occurring phospholipids near physiological pH.