Objective: To explore the functional role of vacuolar H⁺-ATPase in the pH regulation of epididymal fluid and its effect on sperm motility. Design: Experimental study. Setting: Physiology laboratory in a university. Animal(s): Immature male Sprague-Dawley rats. Intervention(s): The H⁺-ATPase inhibitor was applied to the primary culture of epididymal cells. Main Outcome Measure(s): The intracellular luminal fluid pH and sperm percent motility were recorded. Result(s): Double immunofluorescence of H⁺-ATPase and carbonic anhydrase II in primary culture of cauda epididymal epithelial cells showed that the system was a suitable model for investigation of acid secretion by clear cells. Clear cells were pharmacologically distinct from principal cells in acid/base transportation. The intracellular pH recovery from cellular acidification was suppressed by the H⁺-ATPase inhibitor bafilomycin A1(100 nM) and the Na⁺/H⁺ exchanger inhibitor amiloride (1 mM) by 85% and 54%, respectively. These results suggest that, in addition to Na⁺/H⁺ exchanger, clear cells actively pump proton from cytoplasm into extracellular space through H⁺-ATPase. In addition, inhibition of H⁺-ATPase by bafilomycin A1 blocked the acidification of luminal fluid with IC₅₀ values of 12 nM, which supports that H⁺-ATPase acidifies the luminal fluid. We also confirm that the acid fluid regulates rat cauda sperm motility. Conclusion(s): The present work shows that clear cells, the minority cell type of epididymal cell population, play an important role in the pH regulation of epididymal fluid by H⁺-ATPase. © 2010 American Society for Reproductive Medicine.