The genome of Chlamydia trachomatis, one of the most prominent human pathogens, contains two structural genes coding for proteins, herein called Npt1Ct and Npt2Ct (nucleoside phosphate transporters 1 and 2 of C. trachomatis), exhibiting 68 and 61% similarity, respectively, to the ATP/ADP transporter from the intracellular bacterium Rickettsia prowazekii at the deduced amino acid level. Hydropathy analysis and sequence alignments suggested that both proteins have 12 transmembrane domains. The putative transporters were expressed as histidine-tagged proteins in Escherichia coli to study their biochemical properties. His10-Npt1Ct catalyzed ATP and ADP transport in an exchange mode. The apparent Km values were 48 (ATP) and 39 (ADP) microM. ATP and ADP transport was specific since AMP, GTP, CTP, UTP, dATP, dCTP, dGTP, and dTTP did not inhibit uptake. In contrast, His10-Npt2Ct transported all four ribonucleoside triphosphates with apparent Km values of 31 microM (GTP), 302 microM (UTP), 528 microM (CTP), and 1,158 microM (ATP). Ribonucleoside di- and monophosphates and deoxyribonucleotides were not substrates. The protonophore m-chlorocarbonylcyanide phenylhydrazone abolished uptake of all nucleoside triphosphates by Npt2Ct. This observation indicated that His10-Npt2Ct acts as a nucleosidetriphosphate/H+ symporter energized by the proton motive force across the Escherichia coli cytoplasmic membrane. We conclude that Npt1Ct provides chlamydiae with energy whereas Npt2Ct catalyzes the net uptake of ribonucleoside triphosphates required for anabolic reactions.