We have purified a plastidic phosphate transport protein from maize endosperm membranes and cloned and sequenced the corresponding cDNAs from maize endosperm, maize roots, cauliflower buds, tobacco leaves, and Arabidopsis leaves. All of these cDNAs exhibit high homology to each other but only approximately 30% identity to the known chloroplast triose phosphate/phosphate translocators. The corresponding genes are expressed in both photosynthetically active tissues and in nongreen tissues, although transcripts were more abundant in nongreen tissues. Expression of the coding region in transformed yeast cells and subsequent transport measurements of the purified recombinant translocator showed that the protein mediates transport of inorganic phosphate in exchange with C3 compounds phosphorylated at C-atom 2, particularly phosphoenolpyruvate, which is required inside the plastids for the synthesis of, for example, aromatic amino acids. This plastidic phosphate transporter is thus different in structure and function from the known triose phosphate/phosphate translocator. We propose that plastids contain various phosphate translocators with overlapping substrate specificities to ensure an efficient supply of plastids with a single substrate, even in the presence of other phosphorylated metabolites.