The coupling of 4-aminopyrazolo [3,4-d]pyrimidine with the appropriate thio sugar gave a 3:1 ratio of α,β blocked 4-amino-1-(2-deoxy-4-thio-D- erythropentofuranosyl)-1H pyrazolo[3,4-d]pyrimidine nucleosides. The mixture was deblocked, both the anomers were separated, and the β-anomer was readily deaminated by adenosine deaminase. The nucleosides have been characterized, and their anomeric configurations have been determined by proton NMR. All three nucleosides were evaluated against a panel of human tumor cell lines for cytotoxicity in vitro. The details of a convenient and high yielding synthesis of these nucleosides are described. Copyright © Taylor & Francis, Inc.