Pre-spliceosomes, formed in HeLa nuclear extracts and isolated by sedimentation on glycerol gradients, were chased into spliceosomes, the macromolecular enzyme that catalyzes intron removal. We demonstrate that the pre-spliceosome to spliceosome transition was dependent on ATP hydrolysis and required both a U-rich small nuclear ribonucleoprotein (U snRNP)-containing fraction and a fraction of non-snRNP factors. The active components in the non-snRNP fraction were identified as SR proteins and were purified to apparent homogeneity. Recombinant SR proteins (ASF, SC35, SRp55), as well as gel-purified SR proteins, with the exception of SRp20, were able to restore efficient spliceosome formation. We also demonstrate that the pre-spliceosome to spliceosome transition requires phosphorylated SR proteins. This is the first evidence that SR proteins are required for the pre-spliceosome to spliceosome transition, the step at which the U4/U6.U5 tri-snRNP assembles on the pre-mRNA. The results shown here, together with previous data, suggest U snRNPs require SR proteins as escorts to enter the assembling spliceosome.