Two different experimental approaches have provided evidence that both U2 and Ul snRNPs function in premRNA splicing. When the U2 snRNPs in a nuclear extract are selectively degraded using ribonuclease H and either of two deoxyoligonucleotides complementary to U2 RNA, splicing activity is abolished. Mixing an extract in which U2 has been degraded with one in which Ul has been degraded recovers activity. Use of anti-(U2) RNP autoantibodies demonstrates that U2 snRNPs associate with the precursor RNA during in vitro splicing. At 60 min, but not at 0 min, into the reaction intron fragments that include the branch-point sequence are immunoprecipitated by anti-(U2) RNP At all times, Ul snRNPs bind the 5’splice site of the premRNA. Possible interactionsof the U2 snRNP with the Ul snRNP and with the pre-mRNA during splicing are considered.