Polyclonal antibodies have been raised against a C-terminal peptide of NaPi-1, a recently cloned Na-P_i cotransport system of rabbit kidney cortex with a predicted (unglycosylated) molecular mass of 52 kDa. By Western blot analysis using brush-border membranes isolated from rabbit kidney cortex, two proteins with apparent molecular masses of 64 kDa and 35 kDa were specifically recognized (peptide protectable) by the antiserum obtained. The 64-kDa protein was found to migrate in parallel with the luminal membrane during separation by free-flow electrophoresis of brush-border and basolateral membranes. In immunofluorescence studies using cryostat sections of rabbit kidney, specific binding of antibodies was observed in proximal tubules (including S₁, S₂ and S₃ segments) of superficial and deep nephrons. Anti-(NaPi-1)-antibody-mediated fluorescence was restricted to the brush border of proximal tubular cells. No specific immunoreaction was observed in other tubular segments. The results suggest that the native NaPi-1-related protein (Na-P_i cotransport system) has an apparent molecular mass of 64 kDa and is uniformly expressed in the apical membrane of proximal tubules of all nephron generations in the rabbit kidney. Immunohistochemical localization of the Na-P_i cotransport system NaPi-1 confirms the segmental localization within the nephron of NaPi-1-related mRNA as revealed by the reverse transcriptase/polymerase chain reaction (see preceding paper).