Abstract Adenosine kinase, purified 175-fold from human tumor cells in culture, was free of adenosine deaminase and adenosine monophosphate kinase activities. A nucleoside triphosphate was required for the phosphorylation of adenosine or of 6-methylthiopurine ribonucleoside. Either adenosine triphosphate, inosine triphosphate, or guanosine triphosphate satisfied this requirement, and dATP substituted to a smaller extent. The phosphorylation of adenosine required no added divalent cation, but Mg++ or Mn++ greatly stimulated the phosphorylation of 6-methylthiopurine ribonucleoside; Ca++ or Co++ were less effective in this regard. The pH optima were 6.2 to 6.8 for adenosine as substrate and 6.8 for 6-methylthiopurine ribonucleoside; the Km for adenosme was 1.8 x 10-6 m, and that for 6-methylthiopurine ribonucleoside was 5.0 x 10-5 m. The enzyme did not catalyze phosphorylation of inosine or guanosine; but under the conditions used, it phosphorylated several derivatives and analogues of adenosine to a greater extent than adenosine itself. The well phosphorylated compounds included analogues of adenosine in which (a) -H, -Cl, -SCH3, -OCH3, -NHCH3, -N(CH3)2, or -CH3 replaced the amino group; (b) pyrazole, pyrrole, or triazole rings replaced the imidazole ring; (c) -CH3 or =O substituted for -H in position 1; and (d) -F substituted for-H in position 2. Replacement of the ribofuranosyl group by arabinofuranosyl, xylofuranosyl, or deoxyribosyl groups resulted in compounds that were either poorly or not at all phosphorylated. 6-Mercaptopurine ribonucleoside, 6,6'-dithiobis(9-β-d-ribofuranosylpurine), and 2-fluoro-6-mercaptopurine ribonucleoside, compounds which were not themselves phosphorylated, inhibited the phosphorylation of both adenosine and 6-methylthiopurine ribonucleoside.