The first AG dinucleotide downstream from the branchpoint sequence (BPS) is chosen as the 3' splice site during catalytic step II of the splicing reaction. The mechanism and factors involved in selection of this AG are not known. Early in mammalian spliceosome assembly, U2AF65 binds to the pyrimidine tract between the BPS and AG. Here we show that U2AF65 crosslinking is replaced by crosslinking of three proteins of 110, 116 and 220 kDa prior to catalytic step II, and we provide evidence that all three proteins are components of U5 snRNP. These proteins interact with pre-mRNA in the region spanning from immediately downstream of U2 snRNP's binding site at the BPS to just beyond the 3' splice site. We also demonstrate that there are strict constraints on both the sequence and the distance between the BPS and AG for catalytic step II. Together, these observations suggest that U5 snRNP is positioned on the 3' splice site by an interaction (direct or indirect) with U2 snRNP bound at the BPS and by a direct interaction with the pyrimidine tract. The functional AG for catalytic step II may be specified, in turn, by its location with respect to the U5 snRNP binding site.