An Arabidopsis genomic sequence was recently shown to share similarity with bacterial and eukaryotic phosphate (Pi) transporters. We have cloned the corresponding cDNA, which we named Pht2;1, and subsequently performed gene expression studies and functional analysis of the protein product. The cDNA encodes a 61-kD protein with a putative topology of 12 transmembrane (TM) domains interrupted by a large hydrophilic loop between TM8 and TM9, Two boxes of eight and nine amino acids, located in the N- and C-terminal domains, respectively, are highly conserved among species across all kingdoms (eubacteria, archea, fungi, plants, and animals). The Pht2;1 gene is predominantly expressed in green tissue, the amount of transcript staying constant in leaves irrespective of the Pi status of the shoot; in roots, however, there is a marginal increase in mRNA amounts in response to Pi deprivation. Although the protein is highly similar to eukaryotic sodium-dependent Pi transporters, functional analysis of the Pht2;1 protein in mutant yeast cells indicates that it is a proton/Pi symporter dependent on the electrochemical gradient across the plasma membrane. Its fairly high apparent K-m for Pi (0.4 mM) and high mRNA content in the shoot, especially in leaves, suggest a role for shoot organs in Pi loading. Pht2;1 thus differs from members of the recently described plant Pi transporter family in primary structure, affinity for Pi, and presumed function.