The transport of phosphate (Pi) between subcellular compartments is central to metabolic regulation. Although some of the transporters involved in controlling the intracellular distribution of Pi have been identified in plants, others are predicted from genetic and biochemical studies. The Arabidopsis thaliana genome encodes a family of six proteins that share similarity with SLC17/type I Pi transporters, a diverse group of animal proteins involved in the transport of Pi, organic anions and chloride. Heterologous expression in yeast, and gene expression and localization studies in plants were used to characterize all six members of this Arabidopsis family, which we have named PHT4. All of the PHT4 proteins mediate Pi transport in yeast with high specificity. Bioinformatic analysis and localization of PHT4-GFP fusion proteins indicate that five of the proteins are targeted to the plastid inner envelope membrane, and the sixth resides in the Golgi apparatus. PHT4 genes are expressed in both roots and leaves although two of the genes are expressed predominantly in leaves and one mostly in roots. These expression patterns, together with Pi transport activities and subcellular locations, suggest roles for PHT4 proteins in the transport of Pi between the cytosol and chloroplasts, heterotrophic plastids and the Golgi apparatus.