Seven monoclonal antibodies (mAbs) were raised against a rabbit renal brush-border glycoprotein (molecular mass, 63-66 kDa), presumably involved in Na+/Pi cotransport, which we had previously purified and reconstituted in active form in proteoliposomes (Debiec, H., Lorenc, R., and Ronco, P. M. (1992) Biochem. J. 286, 97-102). Antibody specificity for the 63-66-kDa protein was analyzed by enzyme-linked immunosorbent assay and confirmed by Western blotting and immunoaffinity chromatography of solubilized brush-border membranes (BBM), which both yielded a single 63-66-kDa band. Enzyme-linked immunosorbent assay and immunoblotting of renal cortical cell subfractions localized the immunoreactive protein to the brush-border membrane. This location was confirmed by indirect immunofluorescence of kidney cortex sections. Binding of two of the seven mAbs (63A20 and 206A126) to native BBM only occurred when the related epitope was exposed in the presence or absence of Na+, respectively; the other mAbs did not react with native BBM probably because of intramembranous orientation of the epitopes. mAb 63A20 inhibited dose-dependently Na+/Pi cotransport when preincubation of BBM was carried out in the presence of Na+ but did not affect Na+/D-glucose cotransport. Proteoliposomes formed from BBM proteins depleted of the 63-66-kDa protein by affinity chromatography with mAb 63A20 showed an 85% reduction in Na+/Pi cotransport, whereas Na+/D-glucose cotransport was not modified. These results thus establish that the 63-66-kDa BBM protein is the essential component of the Na+/Pi cotransport system. The present study also provides the first immunologic tools available for immunohistochemical localization of the Na+/Pi cotransporter. Finally, the identification of a functional epitope by mAb 63A20 opens up new ways to explore the molecular aspects of Pi uptake.