Nearest-neighbor recognition experiments, which have been carried out using exchangeable dimers derived from 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and 1-palmitoyl-2-oleoyl-sn-glycerophosphoethanolamine,indicate that replacement of H2O by D2O can significantly influence phospholipid mixing, but only in bilayers that are saturated and devoid of cholesterol. These findings, together with those of previous electron spin resonance spin-labeling studies, indicate that mammalian membranes, which are rich in cholesterol and unsaturated phospholipids, are ideal hydrophobic barriers.