The aromatic acid:H+ symporter family of integral membrane proteins play an important role in the microbial metabolism of aromatic compounds. Here, we show that the 4-hydroxybenzoate transporter from Acinetobacter sp. ADP1, PcaK, can be successfully overexpressed in Escherichia coil and purified by affinity chromatography. Affinity-purified PcaK is a stable, monodisperse homotrimer in the detergent n-dodecyl-beta-D-maltopyranoside supplemented with cholesteryl hemisuccinate. The purified protein has alpha-helical secondary structure and can be reconstituted to a functional state in synthetic proteoliposomes. Asymmetric substrate transport was observed when proteoliposomes were energized by applying an electrochemical proton gradient (Delta(mu) over bar (+)(H)) or a membrane potential (Delta Psi) but not by Delta pH alone. PcaK was selective in transporting 4-hydroxybenzoate and 3,4-dihydroxybenzoate over closely related compounds, confirming previous reports on substrate specificity. However, PcaK also showed an unexpected preference for transporting 2-hydroxybenzoates. These results provide the basis for further detailed studies of the structure and function of this family of transporters. (C) 2014 The Authors. Published by Elsevier Inc.