Unlike autocatalyzed self-splicing of group I or group II introns, the removal of pre-mRNA introns in vitro occurs in the spliceosome. The spliceosome is a multicomponent complex composed of pre-mRNA, small nuclear ribonucleoprotein particles, and protein factors. ATP is required for the assembly of the spliceosome and both transesterification reactions. An RNA-dependent ATPase, the product of the yeast PRP2 gene, has been shown to be involved in the first transesterification of pre-mRNA splicing but not in spliceosome assembly. By using ATP analogs, we show that hydrolysis of ATP, mediated through a PRP2-dependent step, is required for the first catalytic event of pre-mRNA splicing. Furthermore, by using a two-step purification procedure, we have isolated a PRP2-containing spliceosome within which the first transesterification readily occurs after the addition of ATP. No additional macromolecules were required. Our results suggest that PRP2 binds to the spliceosome, interacting with an unidentified RNA species in the spliceosome, hydrolyzing ATP and allowing splicing to proceed. We postulate that PRP2 may function to induce a conformational change within the spliceosome. Alternatively, PRP2 may be involved in a proofreading step prior to splicing.