A cDNA library from rabbit kidney cortex was screened for expression of Na-dependent transport of phosphate (Pi) using Xenopus laevis oocytes as an expression system. A single clone was eventually isolated (designated NaPi-1) that stimulated expression of Na/Pi cotransport approximately 700-fold compared to total mRNA. The predicted sequence of the Na/Pi cotransporter consists of 465 amino acids (relative molecular mass, 51,797); hydropathy profile predictions suggest six (possibly eight) membrane-spanning segments. In vitro translation of NaPi-1/complementary RNA in the presence of pancreatic microsomes indicated NaPi-1 to be a glycosylated protein; four potential N-glycosylation sites are present in the amino acid sequence. Northern blot analysis demonstrated the presence of NaPi-1/mRNA in kidney cortex and liver; no hybridization signal was obtained with mRNA from other tissues (including small intestine). Kinetic analysis of Na/Pi cotransport expressed by NaPi-1/complementary RNA demonstrated characteristics (sodium interaction) similar to those observed in cortical apical membranes. The alignment of 5 amino acid residues (Gly342/Ala381-Xaa-Xaa-Xaa-Xaa-Leu386-Xaa-Xaa-Xaa-P ro390- Arg391) is consistent with a motif proposed for Na-dependent transport systems. We conclude that we have cloned a cDNA for a Na/Pi cotransport system present in rabbit kidney cortex.