Conformational dynamics have been studied in proteins, but the extent of thermal motion within large compact RNA molecules, including ribozymes, has not been systematically explored. Methods are developed for the incorporation of the alkyl-phenyl disulfide 1 and the alkyl thiol 2 at specific ribose 2’-positions within RNA. These groups react under mild conditions to form disulfide cross-links by thiol-disulfide interchange. When incorporated on opposite faces of a short, continuous RNA helix, these reactants do not form a disulfide cross-link. In contrast, in an active 310-nucleotide group I ribozyme prepared by semisynthetic methods, sites separated by a distance of up to 50 Angstrom in the structure are cross-linked at rates only 3- to 15-times slower than cross-linking between proximal sites. Furthermore, ribozymes with normally distant sites held together by a disulfide cross-link retain substantial catalytic activity. These results reveal unexpectedly large thermal motions between domains of a compact folded ribozyme.