We describe a simple subcellular fractionation scheme aimed at separating early endosomes from the plasma membrane in view of studying the possible arrival of plasma membrane-bound toxins, proteins or other extracellular ligands in endosomes. Plasma membrane proteins were labeled with the impermeable reagent sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC) biotin at 4°C. In a separate set of cells, early endosomes were labeled by internalization of horseradish peroxidase from the medium for 5 min. The first step of the purification, which consists of a step sucrose gradient, led to three fractions, respectively: enriched in biosynthetic membranes (interface 3), in plasma membrane and early endosomes (interface 2), and in late endosomes (interface 1). The second step, in which interface 2 was loaded at the bottom of a 17% Percoll gradient, led to the separation of the plasma membrane, including caveolae and cholesterol-glycolipid rafts, from early endosomes. Western blot analysis of the fractions from the Percoll gradient showed that the transferrin receptor, the small GTPases rab5 and Arf6, as well as annexin II were present both at the plasma membrane and in early endosomes, whereas the caveolar marker caveolin, 1co, migrated only with the biotinylated plasma membrane proteins. We used this fractionation procedure to show that the pore-forming toxin aerolysin does not reach the endocytic compartments of baby hamster kidney (BHK) cells. The procedure should be generally useful in rapidly determining whether extracellular proteins or ligands reach endosomes.