Ammonia metabolism and transport are critical for acid-base homeostasis. The ammonia transporter family member Rh C glycoprotein (Rhcg) is expressed in distal renal tubular segments, and its expression is regulated in parallel with renal ammonia metabolism. However, there are inconsistencies in its reported subcellular distribution, with both apical and basolateral Rhcg reported in rat and human kidney and only apical expression in mouse kidney. Because the membrane location of Rhcg is critical for understanding its physiological role, we reassessed mouse Rhcg localization using refined immunolocalization methods. Two antibodies directed against different Rhcg-specific epitopes identified both apical and basolateral Rhcg immunolabel in mouse kidney. Immunogold electron microscopy both confirmed basolateral plasma membrane Rhcg expression and showed that apical immunolabel represented expression in both the apical plasma membrane and in subapical cytoplasmic vesicles. Immunoblots and Northern blots identified similar bands in Balb/c and C57BL/6 kidneys, suggesting basolateral Rhcg may result from alternative trafficking. Basolateral Rhcg intensity was strain dependent, with less basolateral Rhcg expression in the Balb/c mouse compared with the C57BL/6 mouse. In mice with collecting duct-specific Rhcg gene deletion, generated using Cre-loxP techniques, neither apical nor basolateral Rhcg immunolabel was identified in the collecting duct, confirming that basolateral Rhcg was the product of the same gene product as apical Rhcg. Although basolateral Rhcg expression differed between C57BL/6 and Balb/c mice, Rh B glycoprotein, which is exclusively basolateral, was expressed at similar levels in the two strains. We conclude that Rhcg is present in both the apical and basolateral plasma membrane in the mouse kidney, where it is likely to contribute to renal ammonia metabolism.