Axenically grown Entamoeba histolytica was found to contain adenosine kinase. This organism lacks de novo purine biosynthetic pathways. Adenosine kinase provides the amoeba with a method for salvaging adenosine from ingested nucleosides or from degraded nucleotides. Adenosine kinase was purified 64-fold, by chromatography on Sephacryl S-200, DEAE-cellulose, and (C-8)-adenosine-agarose. The latter separated it from amebal adenylate kinase. Adenosine kinase has a molecular weight of 38,000 and requires glycerol for stability. It utilizes adenosine triphosphate to phosphorylate adenosine, and 7-deazaadenosine (tubercidin), but adenine 9-β-D-arabinofuranoside (ara-A) is not detectably phosphorylated. It requires Mg++ as a cofactor.