The catalytic activity of RNA enzymes is thought to require divalent metal ions, which are believed to facilitate RNA folding and to play a direct chemical role in the reaction. We have found that the hammerhead, hairpin and VS ribozymes do not require divalent metal ions, their mimics such as [Co(NH3)6]3+, or even monovalent metal ions for efficient self-cleavage. The HDV ribozyme, however, does appear to require divalent metal ions for self-cleavage. For the hammerhead, hairpin and VS ribozymes, very high concentrations of monovalent cations support RNA-cleavage rates similar to or exceeding those observed in standard concentrations of Mg2+. Analysis of all reaction components by inductively coupled plasma-optical emission spectrophotometry (ICPOES) and the use of a variety of chelating agents effectively eliminate the possibility of contaminating divalent and trivalent metal ions in the reactions. For the hairpin ribozyme, fluorescence resonance energy transfer experiments demonstrate that high concentrations of monovalent cations support folding into the catalytically proficient tertiary structure. These results directly demonstrate that metal ions are not obligatory chemical participants in the reactions catalysed by the hammerhead, hairpin, and VS ribozymes. They permit us to suggest that the folded structure of the RNA itself contributes more to the catalytic function than was previously recognised, and that the presence of a relatively dense positive charge, rather than divalent metal ions, is the general fundamental requirement. Whether this charge is required for catalysis per se or simply for RNA folding remains to be determined.