The three-dimensional structure of human erythrocytic purine nucleoside phosphorylase has been determined at 3.2 A resolution using x-ray diffraction data. Intensity data were measured using radiation from the Synchrotron Radiation Source, Daresbury, England, and oscillation film techniques. Phases were determined by using multiple isomorphous replacement methods with four heavy-atom derivatives and were improved using solvent flattening techniques. Purine nucleoside phosphorylase exists in the crystal as a trimer in which subunits are related by a crystallographic 3-fold axis. Each subunit contains an eight-stranded mixed beta-sheet and a five-stranded mixed beta-sheet which join to form a distorted beta-barrel structure. This core beta-structure is flanked by seven alpha-helices in a manner that generates a novel folding pattern. The active site, which was characterized from binding of the substrate analogs 8-iodoguanine and 5'-iodoformycin B, is located near the subunit-subunit boundary within the trimer and involves seven different segments from one subunit and an additional short segment from an adjacent subunit. In the crystal, the phosphate-binding site is probably occupied by a sulfate ion. The specificity of purine nucleoside phosphorylase for guanine, hypoxanthine, and their analogs can be explained on the basis of the arrangement of hydrogen bond donors and acceptors in the active site.