Phenotype analysis has demonstrated that AQP1 null mice are polyuric and manifest a urinary concentrating defect because of an inability to create a hypertonic medullary interstitium. We report here that deletion of AQP1 is also associated with a decrease in urine pH from 6.15±0.1 (SE) to 5.63±0.07 (SE). To explore the mechanism of the decrease in urine pH we examined the expression of H⁺-ATPase in kidneys of AQP1 null mice. There was strong labeling for H⁺-ATPase in intercalated cells and proximal tubule cells in both AQP1 null and wild type mice. Strong H⁺-ATPase immunostaining was also present in the apical plasma membrane of IMCD cells in AQP1 null mice, whereas no H⁺-ATPase labeling was observed in IMCD cells in wild type mice. In addition, there was an increase in the prevalence of type A intercalated cells in the IMCD of AQP1 null mice, suggesting that the deletion of intercalated cells from the IMCD, that normally occurs during postnatal kidney development, was impaired. Western blot analysis of H⁺-ATPase expression in the different regions of the kidney demonstrated a significant increase in H⁺-ATPase protein in the inner medulla of AQP1 null mice compared with wild type mice. There were no changes in H⁺-ATPase expression in the cortex or outer medulla. These results represent the first demonstration of apical H⁺-ATPase immunoreactivity in IMCD cells in vivo and suggest that the decrease in urine pH observed in AQP1 null mice is due to upregulation of H⁺-ATPase in the IMCD. The induction of H⁺-ATPase expression in IMCD cells of AQP1 null mice may be related to their chronically low interstitial osmolality. The challenge will be to identify the molecular signal(s) responsible for the de novo H⁺-ATPase expression.