Primary deuterium kinetic isotope effects (1 degrees DKIE) on (k(cat)/K-GA, M-1 s(-1)) for dianion (X2-) activated hydride transfer from NADL to glycolaldehyde (GA) catalyzed by glycerol-3-phosphate dehydrogenase were determined over a 2100-fold range of enzyme reactivity: (X2-, 1 degrees DKIE); FPO32-, 2.8 +/- 0.1; HPO32-, 2.5 +/- 0.1; SO42-, 2.8 +/- 0.2; HOPO32-, 2.5 +/- 0.1; S2O32-, 2.9 +/- 0.1; unactivated; 2.4 +/- 0.2. Similar 1 degrees DKIEs were determined for kat. The observed 1 degrees DKIEs are essentially independent of changes in enzyme reactivity with changing dianion activator. The results are consistent with (i) fast and reversible ligand binding; (ii) the conclusion that the observed 1 degrees DKIEs are equal to the intrinsic 1 degrees DKIE on hydride transfer from NADL to GA; (iii) similar intrinsic 1 degrees DKIEs on GPDH-catalyzed reduction of the substrate pieces and the whole physiological substrate dihydroxyacetone phosphate. The ground-state binding interactions for different X2- are similar, but there are large differences in the transition state interactions for different X2-. The changes in transition state binding interactions are expressed as changes in k(cat) and are proposed to represent changes in stabilization of the active closed form of GPDH. The 1 degrees DKIEs are much smaller than observed for enzyme-catalyzed hydrogen transfer that occurs mainly by quantum-mechanical tunneling.