We have analyzed the TbAT1 gene, which codes for the P2 adenosine transporter, from Trypanosoma brucei field isolates to investigate a possible link between the presence of mutations in this gene and melarsoprol treatment failure. Of 65 T. b. gambiense isolates analyzed from a focus in north-western Uganda with high treatment failure rates following melarsoprol therapy, 38 had a mutated TbAT1. Unexpectedly, all individual isolates contained the same set of nine mutations in their TbAT1 genes. Of these, five point mutations resulted in amino acid substitutions, one resulted in the deletion of an entire codon, and three were silent point mutations. Eight of these mutations had previously been reported in a laboratory-derived Cymelarsan-resistant T. b. brucei clone. Identical sets of mutations were also found in a drug-resistant T.b.rhodesiense isolate from south-eastern Uganda and in a T.b.gambiense isolate from a relapsing patient from northern Angola. A deletion of the TbAT1 gene was found in a single T. b. gambiense isolate from a relapsing patient from northern Angola. The data presented demonstrate the surprising finding that trypanosomes from individual relapse patients of one area, as well as from geographically distant localities, contain an identical set of point mutations in the transporter gene TbAT1. They further demonstrate that many isolates from relapse patients contained the wild-type TbAT1 genes, suggesting that melarsoprol refractoriness is not solely due to a mutational inactivation of TbAT1.