The removal of noncoding sequences (introns) from eukaryotic precursor mRNA is catalyzed by the spliceosome, a dynamic assembly involving specific and sequential RNA–RNA and RNA–protein interactions. An essential RNA–RNA pairing between the U2 small nuclear (sn)RNA and a complementary consensus sequence of the intron, called the branch site, results in positioning of the 2′OH of an unpaired intron adenosine residue to initiate nucleophilic attack in the first step of splicing. To understand the structural features that facilitate recognition and chemical activity of the branch site, duplexes representing the paired U2 snRNA and intron sequences from Saccharomyces cerevisiae were examined by solution NMR spectroscopy. Oligomers were synthesized with pseudouridine (ψ) at a conserved site on the U2 snRNA strand (opposite an A-A dinucleotide on the intron strand, one of which forms the branch site) and with uridine, the unmodified analog. Data from NMR spectra of nonexchangeable protons demonstrated A-form helical backbone geometry and continuous base stacking throughout the unmodified molecule. Incorporation of ψ at the conserved position, however, was accompanied by marked deviation from helical parameters and an extrahelical orientation for the unpaired adenosine. Incorporation of ψ also stabilized the branch-site interaction, contributing −0.7 kcal/mol to duplex ΔG°37. These findings suggest that the presence of this conserved U2 snRNA pseudouridine induces a change in the structure and stability of the branch-site sequence, and imply that the extrahelical orientation of the branch-site adenosine may facilitate recognition of this base during splicing.