Group I introns catalyze the self-splicing reaction, and their derived ribozymes are frequently used as model systems for the study of RNA folding and catalysis, as well as for the development of non-native catalytic reactions. Utilizing a group I intron-derived ribozyme from Pneumocystis carinii, we previously reported a non-native reaction termed trans excision-splicing (TES). In this reaction, an internal segment of RNA is excised from an RNA substrate, resulting in the covalent reattachment of the flanking regions. TES proceeds through two consecutive phosphotransesterification reactions, which are similar to the reaction steps of self-splicing. One key difference is that TES utilizes the 3′-terminal guanosine of the ribozyme as the first-step nucleophile, whereas self-splicing utilizes an exogenous guanosine. To further aid in our understanding of ribozyme reactions, a kinetic framework for the first reaction step (substrate cleavage) was established. The results demonstrate that the substrate binds to the ribozyme at a rate expected for simple helix formation. In addition, the rate constant for the first step of the TES reaction is more than one order of magnitude lower than the analogous step in self-splicing. Results also suggest that a conformational change, likely similar to that in self-splicing, exists between the two reaction steps of TES. Finally, multiple turnover is curtailed because dissociation of the cleavage product is slower than the rate of chemistry.