The major physicochemical properties of human erythrocytic purine nucleoside phosphorylase (PNPase) have been described. The molecular weight, estimated by ultracentrifugation, molecular sieving and sucrose density gradient centrifugation, ranged from 87 000 to 92 000. Other physical constants of erythrocytic PNPase were: sedimentation coefficent (s20, w), 5.4 S obtained by sedimentation analysis and 5.5 S by the sucrose density gradient procedure; Stokes radius, 38 A; calculated diffusion coefficient (D20, w), 5.7 X 10(-7) cm2 s-1; frictional ration, 1.29; and partial specific volume calculated from amino acid analysis, 0.73 cm3 g-1. The CD spectra of the human erythrocytic and bovine spleen PNPases were almost identical and indicated a very low alpha-helical content. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the molecular weight of the PNPase subunit is 30 000 +/- 500. These results corroborate earlier reports that the native enzyme is a homologous trimer. Comparative studies with crystalline bovine spleen PNPase confirmed that it is also a trimer but is somewhat smaller than the human erythrocytic enzyme with a molecular weight of about 86 000.