Phylogenetic and biochemical studies of RNA have provided a wealth of information regarding secondary structure; however, knowledge of tertiary structure has been more difficult to obtain. In the current work, electrophoretic and hydrodynamic measurements have yielded the global conformations of a self-cleaving "hammerhead" RNA, both prior to and following self-cleavage. The pre- and post-cleavage structures appear to have nearly identical conformations in which the three helices assume a roughly coplanar arrangement with well-defined interhelix angles. Following self-cleavage, one of the three helix stems (stem I) undergoes a slight (< 10 degrees) realignment, although the relative positions of the other two stems remain unchanged. Extension of stem III from (nominally) three base pairs to nine base pairs, known to dramatically enhance the rate of self-cleavage, results in a substantial (approximately 70 degrees) realignment of stems I and II. The current study represents an approach for studying other nonhelical elements in RNA.