Recently, we identified proteins that co-purify with the human spliceosome using mass spectrometry. One of the identified proteins, CDC5L, corresponds to the human homologue of the Schizosaccharomyces pombe CDC5(+) gene product. Here we show that CDC5L is part of a larger multiprotein complex in HeLa nuclear extract that incorporates into the spliceosome in an ATP-dependent step. We also show that this complex is required for the second catalytic step of pre-mRNA splicing. Immunodepletion of the CDC5L complex from HeLa nuclear extract inhibits the formation of pre-mRNA splicing products in vitro but does not prevent spliceosome assembly. The first catalytic step of pre-mRNA splicing is less affected by immunodepleting the complex. The purified CDC5L complex in HeLa nuclear extract restores pre-mRNA splicing activity when added to extracts that have been immunodepleted using anti-CDC5L antibodies. Using mass spectrometry and database searches, the major protein components of the CDC5L complex have been identified. This work reports a first purification and characterization of a functional, human non-snRNA spliceosome subunit containing CDC5L and at least five additional protein factors.