The specificities of phosphate donors and the effects of metal chelating agents and divalent metal ions on NAD kinase activation by phytochrome-far red-absorbing form (Pfr) were examined. ATP was the most efficient phosphorylating agent. Uridine 5'-triphosphate, cytidine 5'-triphosphate (CTP), inosine 5'-triphosphate, and guanosine 5'-triphosphate in this order caused significant phosphorylation in the dark. Under red light, striking photoactivation of NAD kinase was obtained with ATP and subsequently CTP.In the presence of exogenous Mg(2+), which is required for NAD kinase activity, alpha-nitroso-beta-naphthol, cyanide, and dimethylglyoxime, strongly inhibited the activation by red light without affecting the level of NAD kinase in the dark.Of the divalent cations tested with the KCN-treated phytochrome preparation, only Co(2+) was effective for photoactivation of NAD kinase. Even when Mg(2+), an essential component of NAD kinase, was added to the assay system, the further addition of Co(2+) was required for the activation of NAD kinase by Pfr.