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. A fraction containing entrapped fluorescein was isolated from a cortical homogenate after differential centrifugation and Percoll density gradient centrifugation. This fraction has been dubbed heavy endosomes. To our surprise, small-particle flow cytometry techniques demonstrated that heavy endosomes are homogeneous for entrapped fluorescein dextran and the presence of H(+)-adenosinetriphosphatase activity. The abundance of heavy endosomes, combined with the findings that true endosomal populations are identifiable in other renal cortical fractions, led us to test whether heavy endosomes had the attributes of intermicrovillar clefts. First, we tested whether heavy endosomes vesiculate in vivo or in vitro. Vesicle-by-vesicle flow cytometry analysis of uptake of fluorescein dextran added to the homogenate demonstrated that virtually all the vesicles form in vitro (99 +/- 2%, n = 4). Second, the fraction contains markers associated with intermicrovillar clefts: clathrin light chains, actin, glycoprotein gp280, and gp330, the "Heymann antigen." The presence of the brush border enzyme markers gamma-glutamyl transpeptidase and leucine aminopeptidase in > 99% of the heavy endosomes confirms that the vesicles are of apical origin. The activity of the enzymes colocalized with entrapped markers but was tenfold less than in brush-border membrane vesicles. Heavy endosomes isolated from the rat renal cortex vesiculate in vitro and contain several intermicrovillar markers.