The final urine pH is fine-tuned by intercalated cells (IC) along the connecting tubule (CNT) and collecting duct involving the action of vacuolar H⁺-ATPases. Non-type A-IC express the apical Cl^-/HCO₃^- exchanger pendrin and apical and/or basolateral vacuolar H⁺-ATPases containing the B1 subunit isoform. Mutations in B1 subunit in man and mouse cause distal tubular acidosis due to its importance in acid secretion by type A intercalated cells. Here we investigated the role of the B1 subunit in non type-A IC which are activated during metabolic alkalosis and are thought to depend on the activity of vacuolar H⁺-ATPases localized on the basolateral side. Treatment with deoxycorticosterone acetate and NaHCO₃ for 4 days resulted in a more pronounced alkalosis in B1 deficient mice with increased blood bicarbonate, hypokalemia and hypochloremia. Despite severe alkalosis in B1 deficient mice, the abundance of total pendrin expression was reduced, whereas the relative abundance of pendrin expressing cells was increased. H⁺-ATPase activity in ICs was strongly reduced. Moreover, the a4 and A H⁺-ATPases subunits did not associate with the basolateral domain of B1 deficient non A-ICs. Thus, the B1 subunit is required for the formation of complete and functional basolateral H⁺-ATPases complexes. B1 deficient mice excreted also larger urine volume, associated with lower AQP2 water channel abundance and a higher relative frequency of principal cells in CNT. Thus, the B1 H⁺-ATPase subunit is critical for normal non type A IC function and its loss may also affect the function of principal cells.