Abstract An assay suitable for initial velocity studies was developed to measure adenosine kinase (ATP: adenosine 5′-phosphotransferase, EC 126.96.36.199) activity in crude lysates of human erythrocytes. The pH optimum for the reaction was dependent on the ratio of Mg 2+/ATP used in the assay. With a Mg 2+/ATP ratio of 5.0, the pH optimum was 5.1 and only 10% of the maximal activity was retained at pH 7.3. When a Mg 2+/ATP ratio of 0.50 was used, the pH optimum was 6.2, but 70% of the maximal activity was retained at pH 7.3. For assays performed at pH 7.3, the optimal Mg 2+/ATP ratio was 0.50 for ATP concentrations between 0.50 and 2.0 mmol/l. Mg 2+ was required for reaction, presumably to form the physiological substrate MgATP 2−. However, excess free (uncomplexed) Mg 2+ was a strong inhibitor of the enzyme at pH 7.3. At pH 7.3, the K m values for adenosine and MgATP 2− were 0.66 μmol/l and 82 μmol/l respectively.