Endocytotic vesicles (i.e., endosomes) were prepared from rabbit renal cortex following the intravenous injection of horseradish peroxidase. The endosomal population was derived from a 100,000-g pellet and was found at equilibrium in the lightest area of a sucrose density gradient. This population was separate from other organelles as determined by enzyme markers and had a 2.5-fold enrichment of horseradish peroxidase specific activity compared with the homogenate specific activity corrected for soluble horseradish peroxidase activity. The endosomes contain an oligomycin-insensitive, electrogenic H+-translocating ATPase. They also contain an electroneutral Na+-H+ exchanger. This exchanger is not inhibited by amiloride, and lithium is not a substrate for the exchanger. Lithium does inhibit the Na+-H+ exchanger when added prior to the addition of sodium. The Michaelis constant for sodium of the endosomal Na+-H+ exchanger was found to be 10.0 mM. These data indicate that a population of endosomes from rabbit renal cortex contain an electrogenic H+-ATPase and an electroneutral Na+-H+ exchanger and that the exchanger is distinct from the brush-border membrane Na+-H+ antiporter.