The structural and functional characterization of membrane proteins includes assessment of their topology in the bilayer. In the present work, we successfully used an approach based on comparative epitope accessibility. The classical method of detergent permeabilization of fixed cells allowed antibodies to detect epitopes distributed at either side of each cellular membrane by immunofluorescent staining. Instead, freeze-thawing followed by fixation allowed antibodies to cross only the plasma membrane whereas all intracellular membranes remained impermeable. By combining the immunofluorescence results achieved with these two methods for a variety of known membrane proteins, we showed that epitope accessibility could be accurately determined in proteins residing in the plasma membrane or in intracellular compartments, including the endoplasmic reticulum, lysosomes, peroxisomes, different Golgi regions and the nucleus. Freeze-thawing neither changed the expected distribution of each tested protein nor permeabilized intracellular membranes to antibodies. It only permeabilized the plasma membrane. Furthermore, the protocol proved to be efficient in different kinds of cells, which include MDCK and FRT polarized epithelial cells, HeLa cells and fibroblasts. If the complete topology of an integral membrane protein is known, this method would allow to assign an orientation to epitopes recognized by a panel of monoclonal antibodies. It also avoids the use of toxic reagents for permeabilization. Thus, selective permeabilization of the plasma membrane by freeze-thawing provides an inexpensive and reliable method to investigate the topology of membrane proteins as well as the distribution of soluble proteins.