Human purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of ribonucleosides and 2'-deoxyribonucleosides to the free base and (2'-deoxy)ribose-1-phosphate. The crystal structure previously determined at 3.2 Å resolution by multiple isomorphous replacement methods [Ealick, Rule, Carter, Greenhough, Babu, Cook, Habash, Helliwell, Stoeckler, Parks, Chen and Bugg (1990). J. Biol. Chem. 265, 1812-1820] has now been refined at 2.75 Å. One important solvent molecule in the active site is found to be hydrogen bonded to Thr242 and Asn243, a second molecule to the Glu210 side chain (rotated out of the substrate-binding pocket), and a third bridges the hydroxyl of Tyr88 and SO4(290), located in the phosphate-binding subsite. Hydrophobic interactions dominate the structure and many secondary structural elements are held together by hydrophobic clusters. In the low-resolution structure, the active-site residue Lys244 was modeled to be pointing into the active site, and the refined structure revealed that it is pointing away from the active site. Refinement improved the density for residues 244-249; however, loop 250-263 still shows significant disorder in the native structure. Comparison between crystal structures of native and an inhibitor (THDZ) complex reveals that this flexible loop 250-263 is stabilized by the hydrophobic interactions with the bound inhibitor. The refined structure of PNP is structurally homologous to carboxypeptidase A(CPA), an enzyme which cleaves C-terminus peptides in protein degradation. Similarities and differences between the structures of PNP and CPA are discussed.