The mechanism of purine nucleobase transport in procyclic cells of the protozoan parasite Trypanosoma brucei brucei was investigated. Hypoxanthine uptake at 22°C was rapid and saturable, exhibiting an apparent Km, of 9.3±2.0 μM and a Vmax of 4.5±0.8 pmol · (107cells)−1· s−1. All the natural purine nucleobases tested (Km 1.8–7.2 μM), as well as the purine analogues oxypurinol and allopurinol, inhibited hypoxanthine influx in a manner consistent with the presence of a single high-affinity carrier. Nucleosides and pyrimidine nucleobases had little or no effect on hypoxanthine influx. The uptake process was independent of extracellular sodium, but inhibited by ionophores inducing cytosolic acidification (carbonyl cyanide chlorophenylhydrazone, nigericin, valinomycin) or membrane depolarisation (gramicidin) as well as by the adenosine triphosphatase inhibitors N-ethylmaleimide and N, N′-dicyclohexylcarbodiimide. Using the fluorescent dyes bisoxonol and 2′, 7′-bis-(carboxyethyl)-5, 6-carboxy-fluorescein to determine membrane potential and intracellular pH (pHi, the rate of hypoxanthine uptake was shown to be directly proportional to the protonmotive force. Similarly, under alkaline extracellular conditions hypoxanthine uptake was reversibly inhibited alongside a reduction in protonmotive force. In addition, hypoxanthine accelerated the rate of pH, recovery to pH7 after base-loading with NH4Cl, indicative of a proton influx concurrent with hypoxanthine transport. Finally, after pretreatment of cells with N-ethylmaleimide, hypoxanthine induced a slow membrane depolarisation, demonstrating that hypoxanthine transport is electro-genic. These data show that hypoxanthine uptake in T b. brucei procyclic cells is dependent on the protonmotive force, and are consistent with a nucleobase/H+-symporter model for this transporter.