We have isolated three unique NaPi-2-related protein cDNAs (NaPi-2␣, NaPi-2, and NaPi-2␥) from a rat kidney library. NaPi-2␣ cDNA encodes 337 amino acids which have high homology to the N-terminal half of NaPi-2 containing 3 transmembrane domains. NaPi-2 encodes 327 amino acids which are identical to the Nterminal region of NaPi-2 containing 4 transmembrane domains, whereas the 146 amino acids in the C-terminal region are completely different. In contrast, NaPi-2␥ encodes 268 amino acids which are identical to the Cterminal half of NaPi-2. An analysis of phage and cosmid clones indicated that the three related proteins were produced by alternative splicing in the NaPi-2 gene. In a rabbit reticulocyte lysate system, NaPi-2 ␣, , and ␥ were found to be 36, 36, and 29 kDa amino acid polypeptides, respectively. NaPi-2␣ and NaPi-2␥ were glycosylated and revealed to be 45-and 35-kDa proteins, respectively. In isolated brush-border membrane vesicles, an N-terminal antibody was reacted with 45-and 40-kDa, and a C-terminal antibody was reacted with 37-kDa protein.The sizes of these proteins corresponded to those in glycosylated forms.A functional analysis demonstrated that NaPi-2␥ and -2␣ markedly inhibited NaPi-2 activity in Xenopus oocytes. The results suggest that these short isoforms may function as a dominant negative inhibitor of the fulllength transporter.