The dipole potential generating an electric field much stronger than any other type of membrane potential influences a wide array of phenomena, ranging from passive permeation to voltage-dependent conformational changes of membrane proteins. It is generated by the ordered orientation of lipid carbonyl and membrane-attached water dipole moments. Theoretical considerations and indirect experimental evidence obtained in model membranes suggest that the dipole potential is larger in liquid-ordered domains believed to correspond to lipid rafts in cell membranes. Using three different dipole potential-sensitive fluorophores and four different labeling approaches of raft and nonraft domains, we showed that the dipole potential is indeed stronger in lipid rafts than in the rest of the membrane. The magnitude of this difference is similar to that observed between the dipole potential in control and sphingolipid-enriched cells characteristic of Gaucher's disease. The results established that the heterogeneity of the dipole potential in living cell membranes is correlated with lipid rafts and imply that alterations in the lipid composition of the cell membrane in human diseases can lead to substantial changes in the dipole potential.