In the presence of inhibitors for mitochondrial H+-ATPase, (Na+ + K+)- and Ca2+-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane ("ecto-ATPases"). These ATPases accept several nucleotides, are stimulated by Ca2+ and Mg2+, and are inhibited by N.N'-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brush-border and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg2+ and Mn2+, but not by Ca2+, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator. ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H+ secretion is electrogenic and requires either exit of a permeant anion (Cl-) or entry of a cation, e.g., Na+ via electrogenic Na+/D-glucose and Na+/L-phenylalanine uptake. In the presence of Na+, ATP-driven H+ efflux is stimulated by blocking the Na+/H+ exchanger with amiloride. These data prove the coexistence of Na+-coupled substrate transporters, Na+/H+ exchanger, and an ATP-driven H+ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H+ pumps with (a part of) the DCCD- and NEM- sensitive ATPases at the cytosolic side of the brush-border membrane.