The origins of divalent metal-dependent catalytic properties in phosphoryl transfer by EcoRV endonuclease have been investigated by transient kinetic methods. Pre-steady-state measurements on short oligodeoxynucleotide substrates reveal a burst of product formation for both Mg2+- and Mn2+-catalyzed DNA cleavage reactions, indicating that fur each metal ion the product release step is partially or completely rate-limiting. However, the steepness of the burst is far greater for Mn2+ reactions, and analysis of the steady-state portions of the reaction profiles shows that the overall rate is 6-fold slower in the presence of this cofactor. The strongly rate-limiting product release step in Mn2+ reactions may arise from the higher intrinsic affinity of this metal ion for phosphates. Single-turnover experiments carried out with enzyme in molar excess over DNA were also used to isolate the chemical step of the reaction. In contrast to the slower steady-state rates, both these measurements and the pre-steady-state reaction bursts show that the bond-breaking and bond-making steps are significantly better catalyzed by Mn2+. This supports models for catalysis deduced from X-ray crystal structures of the enzyme-substrate DNA complex, in which a divalent metal ion is directly ligated to the pro-S-P oxygen of the scissile phosphate group.