Recent reports have demonstrated that trans-splicing ribozymes can be employed to repair mutant RNAs. One key factor that influences RNA repair efficiency is the accessibility of the substrate RNA for ribozyme binding, which is complicated by the fact that RNAs may assume multiple conformations and have proteins bound to them in vivo. Here we describe a strategy to map accessible sites on sickle beta-globin (beta(s)-globin) transcripts in vitro and in vivo and to use this information to enhance RNA repair efficiency. Two sites upstream of the sickle mutation were identified as accessible in some fraction of the beta-globin RNA by mapping with a ribozyme library and the accessibility of those sites was assessed by in vitro cleavage analyses. Ribozymes targeting either site could only convert a certain fraction of the beta(s)-globin RNA to product but not drive the reaction to completion. However, cleavage and splicing reactions were driven further toward completion when the two ribozymes were both added to the reactions, suggesting that the substrate RNA is present in multiple conformations in vitro. These two ribozymes were each able to repair beta(s)-globin transcripts in erythrocyte precursors derived from peripheral blood from individuals with sickle cell disease. Moreover, the relative accessibility of the targeted sites in vivo is as predicted by mapping and in vitro analyses. These results demonstrate that this novel RNA mapping strategy represents an effective means to determine the accessible regions of target RNAs and that combinations of trans-splicing ribozymes can be employed to enhance RNA repair efficiency of clinically relevant transcripts such as beta(s)-globin RNA.