Abstract Glycerate-3-kinase (EC 126.96.36.199) from spinach leaves shows absolute specificity for d-glycerate as phosphate acceptor, yielding 3-phosphoglycerate as a product. ATP complexed with either Mg 2+ or Mn 2+ is the preferred phosphate donor. The enzyme has K m (D-glycerate) = 0.25 mM, K m (Mg-ATP) = 0.21 mM, V max = 300 μmol min −1 mg protein −1, and a turnover number = 12,000·min −1. The equilibrium constant for the reaction is approximately 300 at pH 7.8. Pyrophosphate, 3-phosphoglycerate and ribulose 1,5-bisphosphate are the strongest inhibitors among the phosphorylated and nonphosphorylated metabolites tested; however, their regulatory role in vivo is questioned. Substrate kinetics, as well as product and analog inhibition data, are consistent with a sequential random mechanism. The distinct characteristic of the glycerate kinase-catalyzed reaction is the formation of a dead-end complex between the enzyme, d-glycerate, and 3-phosphoglycerate.