Although potential biotechnology applications exist for DNA/RNAzymes (catalytic DNA/RNA or deoxy/ribozymes), few studies have addressed the structure of metal ion binding sites or the metal-dependent activation of these systems. DNA/RNAzymes are sequences of nucleic acids combining catalysis of diverse biochemical reactions with high substrate and cofactor specificity. DNA/RNAzymes are metalloenzymes, requiring metal ions for structural stabilization and divalent metal ions for optimal activity. Through the combinatorial biology technique in vitro selection, libraries as large as ~10¹⁵ sequences may be screened for RNA-cleaving DNAzymes that are dependent on metal ions or small molecules cofactors. In vitro selection has yielded families of Co²⁺-depenent DNAzymes showing high activity and interesting metal ion specificity. A four base difference between the clones 11 and 18 sequences drastically alters the Co²⁺-specifcity, providing an ideal system to study structure function relationships in DNA/RNAzymes. Mutational analysis, artificial phylogenetic analysis and stepwise truncation demonstrate the dependence of Co²⁺-specificity on discrete secondary structure elements, peripheral sequence elements and metastable structures. A unique “2-AT” secondary structural element is important for Co²⁺ specificity. The architecture of the clone 11 DNAzyme utilizes structural strategies similar to the hammerhead, group I intron and Varkud satellite RNAzymes, the HIV genome and human telomerase. The specificity of clone 11 DNAzymes are well suited for spectroscopic characterization of DNA metal binding sites and the development of DNAzyme biosensors and therapeutics. Finally, the development of DNA/RNAzyme-based therapeutics and biosensors must take into account the effects of peripheral sequences and metastable structures to preserve optimal cofactor and substrate specificity.