Using a method of in vitro selection for histidine-dependent cleavage of RNA by DNA enzymes, two different types of catalysts were obtained: One type depends strongly on Mg2+ or other divalent metal ions, the other type performs cleavage reactions independent of any divalent metal ion. This type shows a single conserved region in all clones sequenced, but a common secondary structure is hardly obvious. One of the metal dependent catalysts has an unexpected preference for Ca2+ instead of Mg2+. This catalyst is able to perform the cleavage reaction in trans and shows the properties of a real enzyme. Comparison of the secondary structure and reactivity of this catalyst with Mg2+ and Ca2+ suggests that here a special binding pocket for Ca2+ was selected.