Adenosine transport at 22 degrees C in procyclic forms of Trypanosoma brucei brucei was investigated using an oil-inhibitor stop procedure for determining initial rates of adenosine uptake in suspended cells. Adenosine influx was mediated by a single high affinity transporter (Km 0.26 +/- 0.02 microM, Vmax 0.63 +/- 0.18 pmol/10(7) cells s-1). Purine nucleosides, with the exception of tubercidin (7-deazaadenosine), and dipyridamole inhibited adenosine influx (Ki 0.18-5.2 microM). Purine nucleobases and pyrimidine nucleosides and nucleobases had no effect on adenosine transport. This specificity of the transporter appears to be similar to the previously described P1 adenosine transporter in bloodstream forms of trypanosomes. Uptake of adenosine was Na+-independent, but ionophores reducing the membrane potential and/or the transmembrane proton gradient (monitored with the fluorescent probes bis-(1,3-diethylthiobarbituric acid)-trimethine oxonol and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester, respectively) inhibited adenosine transport. Similarly, an increase in extracellular pH from 7.3 to 8.0 reduced adenosine influx by 30%. A linear correlation was demonstrated between the rate of adenosine transport and the protonmotive force. Adenosine uptake was accompanied by a proton influx in base-loaded cells and was also shown to be electrogenic. These combined results indicate that transport of adenosine in T. brucei brucei procyclics is protonmotive force-driven and strongly suggest that the adenosine transporter functions as an H+ symporter.