It is possible to evolve RNA enzymes in a continuous manner by employing a simple serial-transfer procedure. This method was previously applied only to descendants of one unusually fast-reacting RNA enzyme with RNA ligase activity. The present study establishes a second continuously evolving RNA enzyme, also with RNA ligase activity, but with a completely independent evolutionary origin. Critical to achieving the fast catalytic rate necessary for continuous evolution, development of this enzyme entailed the addition and evolutionary maturation of a 35-nucleotide accessory domain and the application of highly stringent selection pressure, with reaction times as short as 15 ms. Once established, continuous evolution was carried out for 80 successive transfers, maintaining the population against an overall dilution of 10(207)-fold. The resulting RNA enzymes exhibited approximately 10(5)-fold improvement in catalytic efficiency, compared with the starting molecules, and became dependent on a structural feature of the substrate that previously conferred no selective advantage. This adaptation was eliminated by deleting the substrate feature and then carrying out 20 additional transfers of continuous evolution, which resulted in molecules with even greater catalytic activity. Now that two distinct species of continuously evolving enzymes have been established, it is possible to conduct molecular ecology experiments in which the two are made to compete for limited resources within a common environment.