NAD kinase was exclusively found in the soluble portion of the cell components. The enzyme was purified about 100-fold from sucrose extracts of spinach leaves.Plant NAD kinase catalyzes formation of NADP from NAD, ATP, and Mg(++), but scarcely any formation of NADPH from NADH. NADH was a very potent competitive inhibitor of NAD phosphorylation by plant NAD kinase. The K(i) was 1.0 x 10(-4)m.The concentration of Mg(++) required to produce maximal activity was 10(-2)m. Co(++) or Mn(++) could replace Mg(++) in the system.The pH optimum was 6.8. The K(m) for NAD was 2.0 x 10(-3)m and that for ATP was 1.1 x 10(-3)m. No convincing demonstration of the reversibility of the reaction was obtained.It was inferred from above properties of the enzyme that NADP formation ceases in plant tissues in which reduced NAD is accumulated, or in anaerobic tissues.