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Purine nucleoside and nucleobase transporters play a vital role in the metabolism and survival of Trypanosoma brucei because this parasitic protozoan is unable to synthesize purines de novo and thus must acquire preformed purines from its hosts. These parasites express a variety of nucleoside and nucleobase permeases with diverse substrate specificities and distinct patterns of expression during the trypanosome life cycle. We report here that expression of the newly characterized T. brucei nucleoside transporter 10 gene (TbNT10) is up-regulated in the short stumpy form of the life cycle, the bloodstream form of the parasite that is pre-adapted for infection of the tsetse fly vector. Functional expression of TbNT10 in Saccharomyces cerevisiae reveals that the TbNT10 gene encodes an adenosine/guanosine/inosine transporter with apparent Km values of approximately 1 microM and hence is a high affinity purine nucleoside transporter. The restricted expression of TbNT10 during the life cycle suggests that the functional properties of this permease may be specialized to support development and growth of the differentiated short stumpy form or to promote the transformation of short stumpy to procyclic forms within the insect vector.