To initiate a molecular dissection into the mechanism by which purine transport is up-regulated in Crithidia, genes encoding nucleoside transporters from Crithidia fasciculata were cloned and functionally characterized. Sequence analysis revealed CfNT1 and CfNT2 to be members of the equilibrative nucleoside transporter family, and the genes isolated encompassed polypeptides of 497 and 502 amino acids, respectively, each with 11 predicted membrane-spanning domains. Heterologous expression of CfNT1 cRNA in Xenopus laevis oocytes or CfNT2 in nucleoside transport-deficient Leishmania donovani demonstrated that CfNT1 is a novel high affinity adenosine transporter that also recognizes inosine, hypoxanthine, and pyrimidine nucleosides, while CfNT2 is a high affinity permease specific for inosine and guanosine. Southern blot analysis revealed that CfNT2 is present as a single copy within the C. fasciculata genome. Starvation of parasites for purines increased CfNT2 transport activity by an order of magnitude, although Northern blot analysis indicated CfNT2 transcript levels increased by <2-fold. These data imply that this metabolic adaptation can mainly be ascribed to post-transcriptional events. Conversely, Southern analysis of CfNT1 suggests that it is a member of a highly homologous multi-copy gene family, indicating that adenosine transport by C. fasciculata is more complex than previously thought.