Allopurinol is a hypoxanthine analogue used to treat Leishmania infections that also displays activity against the related parasite Trypanosoma brucei. We have investigated the ease by which resistance to this drug is established in Trypanosoma brucei brucei and correlated this to the mechanisms by which it is accumulated by the parasite. Long-term exposure of procyclic T. b. brucei to 3mM allopurinol did not induce resistance. This appears to be related to the fact that allopurinol was taken up through two distinct nucleobase transporters, H1 and H4, both with high affinity for the drug. The apparent Km for [3H]allopurinol transport by H4 (2.1+/-0.4 microM) was determined by expressing the encoding gene in Saccharomyces cerevisiae. Long-term allopurinol exposure did not change Km (hypoxanthine), Ki (allopurinol), or Vmax values of either H1 or H4 transporters and the cells retained their ability to proliferate with hypoxanthine as sole purine source. This study shows that transport-related resistance to purine antimetabolites is not easily induced in Trypanosoma spp. as long as uptake is mediated by multiple transporters.