Methods for studying pre-mRNA splicing in Xenopus oocytes have been improved to allow simultaneous analysis of the splicing reaction and the formation of splicing complexes in vivo. The number, order of appearance, and dependence on intact U1 and U2 snRNPs of complexes formed in vivo on a pre-mRNA substrate are similar but not identical to those observed in vitro. The migration on native gels of the complexes formed in vivo and in vitro is, however, dissimilar. RNAase H-mediated inhibition of splicing caused by oligonucleotide microinjection can be overcome by coinjection of a gene encoding the U snRNA that is targeted for cleavage. Transcripts from the injected gene complement the defect in splicing by assembling into functionally active U snRNPs. Using this assay, mutant U2 snRNAs have been tested for their ability to function in splicing and in splicing complex formation. The results indicate that much of the U2 snRNA, including regions essential for detectable binding of the U2-specific proteins A' and B", is dispensable for splicing.