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Electron paramagnetic resonance spectroscopy (EPR) was used to locate free Mn2+ in the presence of added ribozyme in a study of the hammerhead model ribozymes' Mn2+ binding properties under dilute solution conditions. The study found that hammerhead ribozyme activity is affected by the presence of a specific set of divalent cation sites, over an NaCl concentration ranging from 0.1 to 1. Specifically, the study revealed that, in 0.1 M NaCl, hammerhead activity is detected only after all four high-affinity Mn2+ sites are occupied and increases with the population of the type 2 sites.