Leishmania major and all other parasitic protozoa are unable to synthesize purines de novo and are therefore reliant upon uptake of preformed purines from their hosts via nucleobase and nucleoside transporters. L. major expresses two nucleobase permeases, NT3 that is a high affinity transporter for purine nucleobases and NT4 that is a low affinity transporter for adenine. nt3(–/–) null mutant promastigotes were unable to replicate in medium containing 10 μM hypoxanthine, guanine, or xanthine and replicated slowly in 10 μM adenine due to residual low affinity uptake of that purine. The NT3 transporter mediated the uptake of the anti-leishmanial drug allopurinol, and the nt3(–/–) mutants were resistant to killing by this drug. Expression of the NT3 permease was profoundly downregulated at the protein but not the mRNA level in stationary phase compared with logarithmic phase promastigotes. The nt4(–/–) null mutant was quantitatively impaired in survival within murine bone marrow-derived macrophages. Extensive efforts to generate an nt3(–/–)/nt4(–/–) dual null mutant were not successful, suggesting that one of the two nucleobase permeases must be retained for robust growth of the parasite. The phenotypes of these null mutants underscore the importance of purine nucleobase transporters in the Leishmania life cycle and pharmacology.