The branch site helix from Saccharomyces cerevisiae with pseudouridine (c) incorporated in a phylogenetically conserved position of U2 snRNA features an extrahelical branch site adenosine (A) that forms a base triple interaction with the minor groove edge of a widely conserved purine U2 strand -pyrimidine intron strand (R U2 -Y intron ) base pair two positions upstream. In these studies, NMR spectra of a duplex in which 2-aminopurine (2ap), a fluorescent analog of adenine lacking the proposed hydrogen bond donor, was substituted for the branch site A, indicated that the substitution does not alter the extrahelical position of the branch site residue; thus, it appears that a hydrogen bond between the adenine amino group and the R-Y pair is not obligatory for stabilization of the extrahelical conformation. In contrast, reversal of the orientation of A U2 -U intron to U U2 -A intron resulted in an intrahelical position for the branch site A or 2ap. Fluorescence intensity of 2ap substituted for the branch site A with the original R U2 -Y intron orientation (AU or GC) was high, consistent with an extrahelical position, whereas fluorescence in helices with the reversed R-Y orientation, or with a mismatched pair (A-U / G d A or U d C), was markedly quenched, implying that the residue was stacked in the helix. The A 59 to the branch site residue was not extrahelical in any of the duplexes. These findings suggest that the R U2 -Y intron base pair orientation in the c-dependent branch site helix plays an important role in positioning the branch site A for recognition and/or function.