While purine transport has been widely studied in protozoa, almost nothing is known about their capacity to salvage pyrimidines. Here, we report a Leishmania major transporter with high affinity for uracil (Km=0.32+/-0.07 microM) which we designated LmU1. This transporter displayed a high degree of specificity, as it had virtually no affinity for cytosine, thymine or purine nucleobases, nor did it transport pyrimidine nucleosides. Highest affinity was for 5-fluorouracil. The results show that the permeant binding site of LmU1 interacts strongly with the keto groups of uracil, as shown by a low affinity for 2-thio- and 4-thiouracil. LmU1 appears to further bind uracil through a weak hydrogen bond with N(1)H of the pyrimidine ring in addition to a stronger H-bond with N(3)H. Substrate binding and selectivity were strikingly similar to that of the U1 transporter in the related kinetoplastid Trypanosoma brucei. Uracil analogues likely to be transported by LmU1 were also screened for antileishmanial activity, with 5-fluorouracil displaying strong activity against promastigotes and intracellular amastigotes. Overall, the results show that, like purine nucleobase transport, pyrimidine nucleobase transport function is very similar in L. major and T. brucei insect forms.