Functional domains within the mammalian U2 snRNP particle that are required for pre-mRNA splicing have been analysed using antisense oligonucleotides. A comparison of the melting temperatures of duplexes formed between RNA and different types of antisense oligonucleotides has demonstrated that the most stable hybrids are formed with probes made of 2′-O-allyl RNA incorporating the modified base 2-aminoadenine. We have therefore used these 2′-O-allyl probes to target sequences within the central domain of U2 snRNA. Overlapping biotinylated 2′-O-allyloligoribonucleotides complementary to the stem loop IIa region of U2 snRNA (nucleotides 54-72) specifically affinity selected U2 snRNA from HeLa nuclear extracts. These probes inhibited mRNA production in an in vitro splicing assay and caused a concomitant accumulation of splicing intermediates. Little or no inhibition of spliceosome assembly and 5′ splice site cleavage was observed for all pre-mRNAs tested, indicating that the oligonucleotides were specifically inhibiting exon ligation. This effect was most striking with a 2′-O-allyloligoribonucleotide complementary to U2 snRNA nucleotides 57-68. These results provide evidence for a functional requirement for U2 snRNP in the splicing mechanism occurring after spliceosome assembly.