The metal ion dependence of Escherichia coli ribonuclease H activity has been examined by monitoring the change in absorbance of nucleotide substrate by rapid (stopped-flow) kinetic methods. Kinetic equations that fully account for enzyme activation, and substrate inhibition at high metal concentration, have been derived. inhibition constants correlate with metal nucleotide binding affinities. Comparison of thermodynamic (Huang, H.-W.; Cowan, J. A fur. J. Biochem. 1994, 219, 253-260) and kinetic data suggests that there is one essential catalytic metal cofactor required for ribonuclease H activation, rather than a binuclear magnesium site. This conclusion is in accord with recent crystallographic data on the Mg2+-bound enzyme (Katayanagi, K.; Okumura, M.; Morikawa, K. Proteins 1993, 17, 337-346). Similar conclusions are likely to hold for the structurally homologous RNase H domains of retroviral reverse transcriptase.