The P9 stem-loop is one of the conserved structural elements found in all group-I introns. Using two deletion mutants in this region of the Tetrahymena thermophilia large ribosomal subunit intron, we show that removal of the P9 element, either alone, or together with the non-conserved downstream P9.1 and P9.2 elements, results in an intron incapable of the first step of the splicing reaction at a low concentration of Mg2+. The mutant introns also require high concentrations of Mg2+ for the second step in splicing, as well as hydrolysis reactions, suggesting that P9, as well as P9.1 and P9.2, are important structural elements in the final folded form of the intron. In addition, RNase-T1-mediated-structure-probing experiments demonstrated that the loss of P9, P9.1 and P9.2 changes the structural context of the region binding the 5' splice site. The deletions lead to less efficient recognition of the 3' splice site and an accumulation of unligated exons. These observations support the view that the P9, P9.1 and P9.2 stem-loops play an important role in the binding of the 3' splice site.