HeLa cell nuclear splicing extracts have been prepared that are specifically and efficiently depleted of U1, U2, or U4 U6 snRNPs by antisense affinity chromatography using biotinylated 2′-OMe RNA oligonucleotides. Removal of each snRNP particle prevents pre-mRNA splicing but arrests spliceosome formation at different stages of assembly. Mixing extracts depleted for different snRNP particles restores formation of functional splicing complexes. Specific binding of factors to the 3′ splice site region is still detected in snRNP-depleted extracts. Depletion of U1 snRNP impairs stable binding of U2 snRNP to the pre-mRNA branch site. This role of U1 snRNP in promoting stable presplicing complex formation is independent of the U1 snRNA-5′ splice site interaction.