Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway and an attractive target for drug design. The crystal structure of Streptococcus mutants purine nucleoside phosphorylase (Smu PNP) has been solved by molecular replacement at 1.80 A resolution and refined to R factors of 19.9%/23.7% (Rcryst/Rfree)-Sequence alignment and structural comparison show that Smu PNP has more similarity with PNPs isolated from human and malarial sources than the bacterial PNPs. The structure complexed with hypoxanthine (HPA) and sulfate ion was solved at 2.24 (A) resolution and refined to R factors of 21.6%/24.1% (Rcryst/Rfree). It is interesting to note that the resulting electron density indicated the product, HP A, presents in the active site although inosine was included in the crystallization mixture with Smu PNP. Asn233 and Glu191 are the important residues for ligand binding and recognition. Comparison with PNPs from different species gives detailed information about binding of small molecules on the active site, which is important for the studies of enzymatic mechanism and rational design of specific inhibitors for PNPs.