Continuous exposure of Trypanosoma evansi bloodstream forms to Cymelarsan in vitro resulted in the induction of resistance to the drug over a period of 4.5 months. Induction of resistance to Cymelarsan was accompanied by increased resistance to both Arsobal and Berenil, but Cymelarsan-resistant trypanosomes remained sensitive to Suramin and Antrycide. Since resistance to arsenical drugs in trypanosomes has been linked to changes in adenosine uptake, the adenosine metabolism in drug-sensitive and drug-resistant clones was measured. The initial rate of [3H]-adenosine uptake was much higher in sensitive trypanosomes than in drug-resistant parasites, but the amount of radiolabel accumulated by each population was similar. Both adenine and inosine inhibited incorporation of [3H]-adenosine in each population, but in quite different ways. Adenine inhibited more than 80% of adenosine incorporation in drug-sensitive trypanosomes but suppressed less than half of this process in the resistant population. In contrast, inosine inhibited only 10-15% of adenosine incorporation in the sensitive parasites but was inhibitory to a much greater extent in resistant trypanosomes. Lysis of drug-sensitive trypanosomes by 1 microM Cymelarsan was inhibited by adenosine, adenine and Berenil but not by inosine. Furthermore, in drug-sensitive trypanosomes, adenosine uptake could be reduced in three stages by the sequential addition of inosine, Berenil and adenine, whereas in drug-resistant parasites a reduction in adenosine uptake was caused only by the addition of inosine and adenine. These observations provide evidence that the acquisition of resistance to Cymelarsan is accompanied by the loss of one of three adenosine transporters in T. evansi. In drug-sensitive trypanosomes, this transporter mediates the entry of adenine and also of Cymelarsan and Berenil into the parasites.