The mammalian splicing factor U2AF(65) binds to the polypyrimidine tract adjacent to the 3’ splice site and promotes assembly of U2 small nuclear ribonucleoprotein on the upstream branch point, an interaction that involves base pairing with U2 small nuclear RNA(snRNA). U2AF(65) contains an RNA binding domain, required for interaction with the polypyrimidine tract, and an arginine-serine-rich (RS) region, required for U2 snRNP recruitment and splicing, Here it is reported that binding of U2AF(65) to the polypyrimidine tract directed the RS domain to contact the branch point and promoted U2 snRNA-branch point base pairing even in the absence of other splicing factors. Analysis of RS domain mutants indicated that the ability ol U2AF(65) to contact the branch point, to promote the U2 snRNA-branch point interaction, and to support splicing are related activities, requiring only a few basic amino acids. Thus, the U2AF(65) RS domain plays a direct role in modulating spliceosomal RNA-RNA interactions.