Epithelial Na⁺ channel (ENaC)-mediated Na⁺ absorption and BK channel-mediated K⁺ secretion in the cortical collecting duct (CCD) are modulated by flow, the latter requiring an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), microtubule integrity, and exocytic insertion of preformed channels into the apical membrane. As axial flow modulates HCO₃⁻ reabsorption in the proximal tubule due to changes in both luminal Na⁺/H⁺ exchanger 3 and H⁺-ATPase activity (Du Z, Yan Q, Duan Y, Weinbaum S, Weinstein AM, Wang T. Am J Physiol Renal Physiol 290: F289–F296, 2006), we sought to test the hypothesis that flow also regulates H⁺-ATPase activity in the CCD. H⁺-ATPase activity was assayed in individually identified cells in microperfused CCDs isolated from New Zealand White rabbits, loaded with the pH-sensitive dye BCECF, and then subjected to an acute intracellular acid load (NH₄Cl prepulse technique). H⁺-ATPase activity was defined as the initial rate of bafilomycin-inhibitable cell pH (pH_i) recovery in the absence of luminal K⁺, bilateral Na⁺, and CO₂/HCO₃⁻, from a nadir pH of ∼6.2. We found that 1) an increase in luminal flow rate from ∼1 to 5 nl·min⁻¹·mm⁻¹ stimulated H⁺-ATPase activity, 2) flow-stimulated H⁺ pumping was Ca²⁺ dependent and required microtubule integrity, and 3) basal and flow-stimulated pH_i recovery was detected in cells that labeled with the apical principal cell marker rhodamine Dolichos biflorus agglutinin as well as cells that did not. We conclude that luminal flow modulates H⁺-ATPase activity in the rabbit CCD and that H⁺-ATPases therein are present in both principal and intercalated cells.