To investigate the properties of the neomycin-hammerhead interaction, inhibition of hammerhead activity was measured as a function of magnesium concentration and pH. The data are consistent with a simple competition between magnesium and neomycin with about five magnesium ions required to displace neomycin from the hammerhead. The pH dependence of the inhibition of hammerhead cleavage by neomycin and two related aminoglycosides was also determined. The data indicate that at least three of the five positively charged ammonium ions present on neomycin are critical for inhibiting hammerhead function. Taken together, these results suggest that the neomycin-hammerhead interaction is mostly ionic in character.