Background/Aims: Total body phosphate homeostasis is regulated by expression of type IIa sodium phosphate cotransporter (NpT2a) in the apical membrane (BBM) of renal proximal tubule cells. NpT2a expression is regulated by dietary phosphate and PTH but the mechanisms for trafficking of the protein are unknown. Based on 2D gel electrophoresis and mass spectroscopy data that changes in dietary phosphate stimulated changes in BBM expression of vacuolar H+-ATPase, we hypothesized that vacuolar H+-ATPase plays a significant role in regulation of NpT2a in opossum kidney (OK) cells, a model for renal proximal tubule transport. Methods: Role of vacuolar H+-ATPase was studied in opossum kidney (OK) cells by examining the effect of inhibition of vacuolar H+-ATPase on Pi uptake and NpT2a expression. Results: Pretreatment of OK cells with bafilomycin A1 and concanamycin A, inhibitors of vacuolar H+-ATPases, blocked high phosphate- and PTH-induced degradation of NpT2a, but had no effect on high phosphate or PTH induced inhibition of sodium-dependent phosphate transport. Exposure of the cells to bafilomycin A1 significantly decreased phosphate transport and apical membrane expression of NpT2a. Treatment with brefeldin A, an inhibitor of Golgi transport, decreased phosphate transport and apical membrane expression of NpT2a while treatment of cells with both brefeldin A and bafilomycin A1 had no additive effect. Conclusion: We conclude that vacuolar H+-ATPase plays a significant role in exocytosis of NpT2a into the apical membrane and in degradation of NpT2a but has no role in endocytosis.