We have used antisense 2′-OMe RNA oligonucleotides carrying four 5′-terminal biotin residues to probe the structure and function of the human U4 U6 snRNP. Nine oligonucleotides, complementary to multiple regions of U4 and U6 snRNAs, bound stably and specifically to U4 U6 snRNP. This allowed for efficient and selective removal of U4 U6 from HeLa cell nuclear extracts. Binding of oligonucleotides to certain snRNA domains inhibited splicing and affected the U4-U6 interaction. Pre-mRNA and splicing products could also be affinity-selected through binding of the oligonucleotides to U4 U6 snRNPs in splicing complexes. The results suggest that U4 snRNP is not released during spliceosome assembly.