We have applied a self-cleavage assay, developed previously by us, to measure EcoRV-DNA binding in solution. Self-cleavage assay monitors only enzymatically competent complexes of the endonuclease. This technique does not have the limitations of more commonly used gel mobility shift assay while providing the same level of sensitivity. We found that the EcoRV has quite unusual kinetics of specific complex formation in the absence of divalent ions that was not reported previously. A significant fraction of the total enzyme, ā¼ 45%, forms enzymatically competent complexes unusually slowly, especially at pH 7.6. This novel result can be explained by a very slow transition between two conformations of the free enzyme in solution. The equilibrium distribution of the slowly and quickly associating protein structures and their exchange kinetics may depend on many parameters including pH, salt, osmolytes, and divalent cations. The observation of at least two kinetics components in association indicates that EcoRV is an allosteric protein with at least two conformations. Allosterism is now recognized as important concept for DNA-protein complexes, offering an additional level of control over binding and activity. We are continuing our investigation into the EcoRV structures responsible for the different kinetic classes of association.