The RNA branch formed during pre-mRNA splicing occurs at a wide variety of sequences (branch sites) in different mammalian pre-mRNAs. U2 small nuclear ribonucleoprotein (snRNP) binds to the pre-mRNA branch site following the interaction of a protein, U2AF, with the 3' splice site/polypyrimidine tract. Here we show that despite the variability of mammalian branch sites, U2 snRNP has a sequence-specific RNA-binding activity. Thus, RNA branch formation is regulated by two sequence-specific interactions: U2AF with the 3' splice site/polypyrimidine tract, and U2 snRNP with the branch site. The affinity of the branch site for U2 snRNP affects the efficiency of spliceosome assembly and splicing.