Ribozymes and deoxyribozymes have potential application in the treatment of human disease. If nucleic acid enzymes are to be employed as therapeutic agents, then they must function with high specificity and efficiency in the cellular environment in targeting disease-related pathways. Ribozymes that occur in nature have not been evolved for this purpose. Recently described techniques of in vitro selection and evolution, which have been instrumental in discovering new nucleic acid catalysts and in adapting natural ribozymes to novel functions, can also play a role in developing RNA and DNA enzymes for therapeutic purposes. Here we discus specific examples in which in vitro evolution has been used to alter the substrate sequence-specificity of a ribozyme, adapt a ribozyme to target a biological macromolecule other than RNA, develop stabilized RNA ligands that bind to a particular protein target, and develop DNA catalysts with potential therapeutic application. We also assess the prospects for future use of in vitro evolution methods to produce more complex nucleic acid catalysts with broader therapeutic utility.