Renal glutamine synthetase catalyzes the reaction of NH4+ with glutamate, forming glutamine and decreasing ammonia available for net acid excretion. The purpose of the current study was to determine glutamine synthetase's specific cellular expression in mouse kidney and its regulation by hypokalemia, a common cause of altered renal ammonia metabolism. Glutamine synthetase mRNA and protein were present in the renal cortex and in both the outer and the inner stripe of outer medulla (OMo and OMi). Immunohistochemistry showed glutamine synthetase expression throughout the entire proximal tubule and in non-proximal tubule cells. Double-immunolabel with cell-specific markers demonstrated glutamine synthetase expression in Type A intercalated cells, non-A, non-B intercalated cells and distal convoluted tubule cells, but not in principal cells, Type B intercalated cells or connecting segment cells. Hypokalemia induced by feeding a nominally K+-free diet for twelve days decreased glutamine synthetase expression throughout the entire proximal tubule and in the distal convoluted tubule and simultaneously increased glutamine synthetase expression in Type A intercalated cells in both the cortical and outer medullary collecting duct. We conclude that glutamine synthetase is widely and specifically expressed in renal epithelial cells, and that the regulation of expression differs in specific cell populations. Glutamine synthetase is likely to mediate an important role in renal ammonia metabolism.