Abstract The Tetrahymena group I ribozyme requires a hierarchical folding process to form its correct three-dimensional structure. Ribozyme activity depends on the catalytic core consisting of two domains, P4–P6 and P3–P7, connected by a triple-helical scaffold. The folding proceeds in the following order: (i) fast folding of the P4–P6 domain, (ii) slow folding of the P3–P7 domain, and (iii) structure rearrangement to form the active ribozyme structure. The third step is believed to directly determine the conformation of the active catalytic domain, but as yet the precise mechanisms remain to be elucidated. To investigate the folding kinetics of this step, we analyzed mutant ribozymes having base substitution(s) in the triple-helical scaffold and found that disruption of the scaffold at A105G results in modest slowing of the P3–P7 folding (1.9-fold) and acceleration of step (iii) by 5.9-fold. These results suggest that disruption or destabilization of the scaffold is a normal component in the formation process of the active structure of the wild type ribozyme.