Caveolin-1, a major protein of cell surface invaginations called caveolae, is currently believed to cycle between the plasma membrane and intracellular compartments via the endocytotic pathway, at least for part of its itinerary. We studied the distribution of caveolin-1 in cell membranes, using ultrathin cryosections and freeze-fracture immunolabeling and found this protein not only in the cytoplasmic leaflet of the plasma membrane, but also in the exoplasmic leaflet of all intracellular membranes. This sidedness implies that caveolin-1 switches from one membrane leaflet to the other somewhere on its way through the cell and rules out the classic mechanism of endocytotic membrane budding and fusion for caveolin-1 intracellular trafficking. Underlying the sidedness of caveolin-1 may be a fundamental, hitherto unrecognized, mechanism by which proteins transit membranes.