As part of a study of the immune defect related to the absence of purine nucleoside phosphorylase, the substrate analogue 6-hydroxy-9-p-aminobenzylpurine was synthesized. This inosine analogue was a competitive inhibitor with an inhibition constant of about 200 muM. Using trichloro-s-triazine, the inhibitor was coupled to Sepharose, producing an efficient, reusable biospecific affinity gel. The gel was used to purify purine nucleoside phosphorylase from human red cells with an 85% yield and a specific activity of 95 mumol/min/mg. The molecular weight of native purine nucleoside phosphorylase was estimated as 90,400 using high pressure liquid chromatography. A subunit molecular weight of 31,600 was established using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, a trimeric structure for the native enzyme is indicated, which is in accordance with the evidence derived from genetic studies.