Joyce's DNA enzyme catalyzes cleavage of RNAs with almost the same efficiency as the hammerhead ribozyme. The cleavage activity of the DNA enzyme was pH dependent, and the logarithm of the cleavage rate increased linearly with pH from pH 6 to pH 9 with a slope of approximately unity. The existence of an apparent solvent isotope effect, with cleavage of RNA by the DNA enzyme in H2O being 4.3 times faster than cleavage in D2O, was in accord with the interpretation that, at a given pH, the concentration of the active species (deprotonated species) is 4.3 times higher in H2O than the concentration in D2O. This leads to the intrinsic isotope effect of unity, demonstrating that no proton transfer occurs in the transition state in reactions catalyzed by the DNA enzyme. Addition of La3+ ions to the Mg2+-background reaction mixture inhibited the DNA enzyme-catalyzed reactions, suggesting the replacement of catalytically and/or structurally important Mg2+ ions by La3+ ions. Similar kinetic features of DNA enzyme mediated cleavage of RNA and of hammerhead ribozyme-mediated cleavage suggest that a very similar catalytic mechanism is used by the two types of enzyme, despite their different compositions.