The endosomal pathway of the rat renal cortex was labeled by intravenous infusion of fluorescent dextran small enough to cross the glomerular ultrafiltration barrier and be taken up by luminal endocytosis in the proximal tubule. Using Percoll gradient centrifugation, we isolated and characterized a previously undetected renal cortical endosomal fraction slightly lighter than basolateral membranes. Assay of entrapped dextran on a vesicle-by-vesicle basis using small-particle flow cytometry techniques demonstrates homogeneity for entrapped dextran. Flow cytometry colocalization of entrapped markers with brush-border enzymes in > 99% of the vesicles and the absence of Na-K-adenosinetriphosphatase (ATPase) suggest both that these vesicles are of apical origin and that apical enzymes traffic into endosomal elements. Furthermore, two glycoproteins derived from intermicrovillar clefts are detectable in this fraction. Ultrastructurally the vesicles are heterogeneous, consisting of multivesicular bodies together with vesicles of diverse size and coating. Populations of vesicles can be cleanly separated from each other and basolateral membranes according to their surface charge by high-resolution free-flow electrophoresis. Multiparameter flow cytometry analysis demonstrates that a more abundant population of smaller vesicles has brisker H(+)-ATPase activity, whereas a less abundant population of larger vesicles has slower H(+)-ATPase activity. In contrast, brush-border membrane vesicles contained no entrapped markers and lacked H(+)-ATPase activity. Analysis of vesicles prepared after addition of dextran to the homogenate confirms that the vesicles form in vivo. Hence a heterogeneous renal cortical endosomal population is of apical origin.