We have used oligonucleotides made of 2′-OMe RNA to analyze the role of separate domains of U2 snRNA in the splicing process. We show that antisense 2′-OMe RNA oligonucleotides bind efficiently and specifically to U2 snRNP and demonstrate that masking of two separate regions of U2 snRNA can inhibit splicing by affecting different steps in the spliceosome assembly pathway. Masking the 5′ terminus of U2 snRNA does not prevent U2 snRNP binding to pre-mRNA but blocks subsequent assembly of a functional spliceosome. By contrast, masking of U2 sequences complementary to the pre-mRNA branch site completely inhibits binding of pre-mRNA. Hybrid formation at the branch site complementary region also triggers a specific change which affects the 5′ terminus of U2 snRNA.