The vacuolar-type H +-ATPase B1 subunit is heav ily expressed in the intercalated cells of the collecting system, where it contributes to H + transport, but has also been described in other segments of the renal tubule. This study aimed to determine the localization of the B1 subunit of the vacuolar-type H +-ATPase in the early distal nephron, encompassing thick ascending limbs (TAL) and distal convoluted tubules (DCT), in human kidney and determine whether the localization differs between rodents and humans. Antibodies directed against the H +-ATPase B1 subunit were used to determine its localization in paraffin-embedded formalin-fixed mouse, rat, and human kidneys by light microscopy and in sections of Lowicryl-embedded rat kidneys by electron microscopy. Abundant H +-ATPase B1 subunit immunoreactivity was observed in the human kidney. As expected, intercalated cells showed the strongest signal, but significant signal was also observed in apical membrane domains of the distal nephron, including TAL, macula densa, and DCT. In mouse and rat, H +-ATPase B1 subunit expression could also be detected in apical membrane domains of these segments. In rat, electron microscopy revealed that the H +-ATPase B1 subunit was located in the apical membrane. Furthermore, the H +-ATPase B1 subunit colocalized with other H +-ATPase subunits in the TAL and DCT. In conclusion, the B1 subunit is expressed in the early distal nephron. The physiological importance of H +-ATPase expression in these segments remains to be delineated in detail. The phenotype of disease-causing mutations in the B1 subunit may also relate to its presence in the TAL and DCT.