Ribonuclease H (Escherichia coli) contains one strong magnesium-binding site, as determined by metal-titration experiments monitored by high field 1H-NMR and also by direct titration calorimetry. Kinetic and thermodynamic parameters were evaluated by 25Mg-NMR and were as follows: dissociation constant Kd, approximately 60 +/- 10 microM; activation free energy delta G*, approximately 49.8 +/- 0.9 kJ; on/off-rate for magnesium binding Kon, approximately 1.8 x 10(8) M-1 s-1, koff, approximately 1.1 x 10(4) s-1; quadrupole coupling constant chi B, 1.2 +/- 0.2 MHz. The dissociation constant was independently determined by standard analysis of 1H chemical shifts in magnesium-titration experiments and by microcalorimetry (Kd approximately 200 +/- 20 microM). Cobalt hexaamine, which also activates RNase H [Jou, R. & Cowan, J. A. (1991) J. Am. Chem. Soc. 113, 6685-6686], appears to bind at the same location as Mg2+(aqueous). Assignments of C2H and C4H protons to specific histidine residues have been made by two-dimensional correlated spectroscopy experiments. Direct 25Mg-NMR pH titrations show that an ionizable residue (pKa approximately 5.8), most likely one of the carboxylates in the active site, influences magnesium binding. On the basis of the magnesium coordination chemistry elucidated herein, recent proposals on active-site chemistry are critically assessed and general physicochemical aspects of magnesium-binding sites on proteins and enzymes are discussed.