Intron-containing pre-mRNAs are retained in the nucleus until they are spliced. This mechanism is essential for proper gene expression. Although the formation of splicing complexes on pre-mRNAs is thought to be responsible for this nuclear retention activity, the details are poorly understood. In mammalian cells, in particular, very little information is available regarding the retention factors. Using a model reporter gene, we show here that U1 snRNP and U2AF but not U2 snRNP are essential for the nuclear retention of pre-mRNAs in mammalian cells, showing that E complex is the major entity responsible for the nuclear retention of pre-mRNAs in mammalian cells. By focusing on factors that bind to the 3'-splice site region, we found that the 65-kD subunit of U2AF (U2AF(65) ) is important for nuclear retention and that its multiple domains have nuclear retention activity per se. We also provide evidence that UAP56, a DExD-box RNA helicase involved in both RNA splicing and export, cooperates with U2AF(65) in exerting nuclear retention activity. Our findings provide new information regarding the pre-mRNA nuclear retention factors in mammalian cells.