The transbilayer diffusion of unlabeled ceramides with different acyl chains (C 6 -Cer, C 10 -Cer, and C 16 -Cer) was investigated in giant unilamellar vesicles (GUVs) and in human erythrocytes. Incorporation of a very small percentage of ceramides (ϳ0.1% of total lipids) to the external leaflet of egg phosphatidylcholine GUVs suffices to trigger a shape change from prolate to pear shape vesicle. By observing the reversibility of this shape change the transmembrane diffusion of lipids was inferred. We found a half-time for unlabeled ceramide flip-flop below 1 min at 37°C. The rapid diffusion of ceramides in a phosphatidylcholine bilayer was confirmed by flip-flop experiments with a spin-labeled ceramide analogue incorporated into large unilamellar vesicles. Shape change experiments were also carried out with human erythrocytes to determine the trans-membrane diffusion of unlabeled ceramides into a biological membrane. Addition of exogenous ceramides to the external leaflet of human erythrocytes did not trigger echinocyte formation immediately as one would anticipate from an asymmetrical accumulation of new amphiphiles in the outer leaflet but only after ϳ15 min of incubation at 20°C in the presence of an excess of ceramide. We interpret these data as being indicative of a rapid ceramide equilibration between both erythrocyte leaflets as indicated also by electron spin resonance spectroscopy with a spin-labeled ceramide. The late appearance of echinocytes could reveal a progressive trapping of a fraction of the ceramide molecules in the outer erythrocytes leaflet. Thus, we cannot exclude the trapping of ceramides into plasma membrane domains.Ceramide is the backbone and an intermediate molecule in the metabolism of sphingolipids. It is a minor lipid component of the plasma membrane of eukaryotic cells and can be generated in vivo by the degradation of sphingomyelin or of gangliosides. Free ceramide was shown to play a role as second messenger for many cellular functions. Its potent biological activity has been extensively reviewed (1, 2). Because enzymes involved in ceramide generation and metabolism are localized in different subcellular compartments, a challenging question remains unanswered: how do such water-insoluble molecules overcome the successive solubility barriers found during traffic between the membranes of different organelles? In addition, the transmembrane orientation of ceramides must be controlled at each step. Ceramide is synthesized on the cytosolic surface of the endoplasmic reticulum and is converted to galactosylceramide in the luminal leaflet of the endoplasmic reticulum. It is not known if this reorientation within the membrane takes place spontaneously or is catalyzed by a protein. The Golgi system also requires regulated ceramide distribution between leaflets as synthesis of glucosylceramide occurs on the cytosolic side and sphingomyelin (SM) 1 on luminal side (3). The transverse diffusion of several sphingolipid derivatives has been measured with spin-labeled and fluorescent analogues ...

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