The lateral organization of cellular membranes is formed by the clustering of specific lipids, such as cholesterol and sphingolipids, into highly condensed domains (termed lipid rafts). Hence such domains are distinct from the remaining membrane by their lipid structure (liquid-ordered vs. -disordered domains). Here, we directly visualize membrane lipid structure of living cells by using two-photon microscopy. In macrophages, liquid-ordered domains are particularly enriched on membrane protrusions (filopodia), adhesion points and cell-cell contacts and cover 10 -15% of the cell surface at 37°C. By deconvoluting the images, we demonstrate the existence of phase separation in vivo. We compare the properties of microscopically visible domains (<1 m 2 ), with those of isolated detergent-resistant membranes and provide evidence that membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis. membrane domains ͉ macrophages T he lipid raft hypothesis proposes that the lateral organization of cellular membranes is based on the presence of distinct, cholesterol-rich, rigid domains (rafts) (1), which are involved in signal transduction (2), protein sorting, and membrane transport (3, 4). Our understanding of lipid structure and the formation of specific lipid domains within membranes, however, is almost exclusively based on model membrane systems (5). Although phase separation of domains of liquid-ordered structure is predicted to exist in cellular membranes (6, 7), direct demonstration using methodologies such as fluorescence quenching has been difficult to apply to living cells (8). The evidence for the existence of lipid rafts in living cells is largely based on measurements of the clustering (9, 10) or diffusion (11, 12) of lipid raft proteins, which are secondary to the lipid organization.In the present study, we labeled living cells with the fluorescent probe 6-acyl-2-dimethylaminonapthalene (Laurdan), which has been previously used to characterize domain formation and phase separation in model membranes using phospholipid mixtures (13-15) or lipid extracted from cellular membranes (16)(17)(18). Laurdan is an environmentally sensitive fluorescence probe that exhibits a 50-nm red shift as membranes undergo phase transition from gel to fluid, due to altered water penetration into the lipid bilayer (19). Its dipole is aligned parallel to the hydrophobic lipid chains in membranes and is located in both bilayers (20). The probe's fluorescence in water is negligible, and it is not influenced spectroscopically by surface modifications such as lipoprotein binding (20,21). The environmentally induced red shift allows the translation of intensity measurements at different wavelengths into lipid packing orders within the membranes of intact and living cells (20,22,23). Generalized polarization (GP), with a correcting factor G for the experimental setup, is defined analogously to fluorescence polarization by measuring the in...