Individual small nuclear ribonucleoproteins (snRNPs) U1, U2, and U4/U6 were removed from nuclear extracts of HeLa cells by antisense affinity depletion. Addition of a highly purified preparation of SR proteins fully restored splicing activity in reactions depleted of U1 snRNP but did not reconstitute splicing in reactions depleted of the other snRNPs. Affinity selection experiments revealed that spliceosomes lacking U1 snRNA formed in the U1 snRNP-depleted reactions reconstituted with SR proteins. Thus, high concentrations of SR proteins facilitate the assembly of precursor messenger RNA (pre-mRNA) into a spliceosome in the absence of interactions with U1 snRNP.