Distinct anion transport processes have been identified in the mammalian renal proximal tubule, but none of the responsible proteins or genes have been isolated. A 43kDa rabbit microvillus membrane protein that is immunologically related to the erythroid anion exchanger (band 3) was a candidate for a renal anion transporter. To examine the structural relationship with band 3, we cloned cDNAs encoding the 43kDa protein. The 43kDa band-3-1ike protein was purified, and a novel sequence of 24 amino acids was obtained from the N-terminus. Degenerate oligonucleotides were synthesized based on this sequence, and the polymerase chain reaction with single-sided specificity was used to amplify and clone a 1330bp cDNA from rabbit renal cortex. Additional overlapping 272bp and 1123bp cDNAs were obtained by synthesizing and screening a rabbit renal cortical cDNA library. The composite sequence was 1483bp, terminated with (A)16, and was similar in size to the principal transcript expressed in rabbit renal cortex. The single long open reading frame was predicted to encode a protein composed of 410 amino acids with a molecular mass of 45193Da; 15 amino acids predicted to reside at the N-terminus were absent in the mature protein and may constitute a signal peptide. There was only limited sequence similarity with human erythroid band 3. Rather, the sequence was highly similar to microsomal dipeptidase, including the presence of a signal peptide and a consensus sequence for covalent linkage to glycosyl-phosphatidylinositol. In summary, the 43kDa protein from rabbit renal cortex that is recognized by a monospecific antibody to erythroid band 3 is most likely a microvillus membrane dipeptidase.