The active site components and substrate specificity of an RNA Diels-Alderase (DA22) were investigated. Diels-Alderase activity was found to be highly dependent on a unique 5-position pyridyl modified uridine. Even closely related pyridyl modifications failed to yield active catalysts. Substrate specificity of this Diels-Alderase was remarkable. Experiments with alternative diene and dienophile substrates showed the active site of the Diels-Alderase to be highly discriminating, even against molecules of similar reactivity and structure. Inhibition studies with a series of product analogues established that the RNA recognizes functional components in and around the reaction center of both the diene and the dienophile. Taken together, these results suggest that DA22 can fold into a structure that produces an intricate metal/ligand dependent active site capable of highly specific molecular recognition.