Discrimination between splice sites and similar, nonsplice sequences is essential for correct intron removal and messenger RNA formation in eukaryotes. The 65- and 35-kD subunits of the splicing factor U2AF, U2AF ⁶⁵ and U2AF ³⁵ , recognize, respectively, the pyrimidine-rich tract and the conserved terminal AG present at metazoan 3′ splice sites. We report that DEK, a chromatin- and RNA-associated protein mutated or overexpressed in certain cancers, enforces 3′ splice site discrimination by U2AF. DEK phosphorylated at serines 19 and 32 associates with U2AF ³⁵ , facilitates the U2AF ³⁵ -AG interaction and prevents binding of U2AF ⁶⁵ to pyrimidine tracts not followed by AG. DEK and its phosphorylation are required for intron removal, but not for splicing complex assembly, which indicates that proofreading of early 3′ splice site recognition influences catalytic activation of the spliceosome.