Splicing of mRNA precursors (pre-mRNAs) comprises a series of ATP-dependent steps, the first of which is the stable binding of U2 snRNP at the pre-mRNA branchpoint. The basis of ATP use for the interaction between U2 snRNP and the branchpoint is unclear, and, in particular, none of the known mammalian factors required for this step have the sequence characteristics of proteins that hydrolyze ATP. Entry of U2 snRNP into the spliceosome is initiated by interaction of the essential splicing factor U2AF65 with the pre-mRNA polypyrimidine tract. In this report we identify a new region of U2AF65 required for function, and use this information to clone a human 56-kD U2AF65 associated protein (UAP56). We show that UAP56 is an essential splicing factor, which is recruited to the pre-mRNA dependent on U2AF65, and is required for the U2 snRNP-branchpoint interaction. The sequence of UAP56 indicates it is a member of the DEAD box family of RNA-dependent ATPases, which mediate ATP hydrolysis during several steps of yeast pre-mRNA splicing. Our results reveal a new function of U2AF65: to position a DEAD box protein required for U2 snRNP binding at the pre-mRNA branchpoint region.