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Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine-salvage pathway, which allows cells to utilize preformed bases and nucleosides in order to synthesize nucleotides. PNP is specific for purine nucleosides in the β-configuration and exhibits a strong preference for purines containing a 6-keto group and ribosyl-containing nucleosides relative to the corresponding analogues. PNP was crystallized in complex with ligands and data collection was performed using synchrotron radiation. This work reports the structure of human PNP in complex with guanosine (at 2.80 Å resolution), 3′-deoxyguanosine (at 2.86 Å resolution) and 8-azaguanine (at 2.85 Å resolution). These structures were compared with the PNP–guanine, PNP–inosine and PNP–immucillin-H complexes solved previously.