Abstract This study establishes a new assay for measuring the transbilayer movement of dehydroergosterol (DHE) in lipid membranes. The assay is based on the rapid extraction of DHE by methyl- β-cyclodextrin (M-CD) from liposomes. The concentration of DHE in the liposomal membrane was measured by using fluorescence resonance energy transfer (FRET) from DHE to dansyl-phosphatidylethanolamine, which is not extracted from liposomes by M-CD. The method was applied to small (SUV) and large (LUV) unilamellar vesicles of different compositions and at various temperatures. From the kinetics of FRET changes upon extraction of DHE from membranes, rates of M-CD mediated extraction and flip-flop of DHE could be deduced and were found to be dependent on the physical state of the lipid phase. For egg phosphocholine and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine in the liquid-crystalline state, halftimes of extraction and transbilayer movement were <5 s and ∼20–50 s, respectively, at 10°C. For 1,2-dimyristoyl- sn-glycero-3-phosphocholine-SUV being in the gel state at 10°C, the respective halftimes were 28 s and 5–8 min. Surprisingly, DHE could not be extracted from LUV consisting of 1,2-dimyristoyl- sn-glycero-3-phosphocholine. This might be an indication of specific interactions between DHE molecules in membranes depending on the phospholipid composition of the membrane.