Much interest has focused on the mechanisms of the five naturally occurring self-cleaving ribozymes, which, in spite of catalyzing the same reaction, adopt divergent strategies. These ribozymes, with the exception of the recently described glmS ribozyme, do not absolutely require divalent metal ions for their catalytic chemistries in vitro. A mechanistic investigation of an in vitro-selected, RNA-cleaving DNA enzyme, the bipartite, which catalyzes the same chemistry as the five natural self-cleaving ribozymes, found a mechanism of significant complexity. The DNAzyme showed a bell-shaped pH profile. A dissection of metal usage indicated the involvement of two catalytically relevant magnesium ions for optimal activity. The DNAzyme was able to utilize manganese(II) as well as magnesium; however, with manganese it appeared to function complexed to either one or two of those cations. Titration with hexaamminecobalt(III) chloride inhibited the activity of the bipartite; this suggests that it is a metalloenzyme that utilizes metal hydroxide as a general base for activation of its nucleophile. Overall, the bipartite DNAzyme appeared to be kinetically distinct not only from the self-cleaving ribozymes but also from other in vitro-selected, RNA-cleaving deoxyribozymes, such as the 8-17, 10-23, and 614.