Intervening sequences are removed from nuclear pre-mRNAs in a well-defined multi-step pathway. Small nuclear ribonucleoprotein particles (snRNPs) and numerous protein factors are essential for the formation of the active spliceosome in which intron excision proceeds in two successive transesterification reactions. Important elements for catalysis are the RNA moieties of the snRNPs that align the pre-mRNA splice sites in the active center of the spliceosome. Although pre-mRNA splicing is almost certainly RNA-mediated, both snRNA-associated proteins and non-snRNP splicing factors participate in each step of the splicing reaction. Splicing proteins exert auxiliary functions in the recognition, selection, and juxtaposition of the splice sites and drive conformational changes during spliceosome assembly and catalysis. Many splicing factors have been isolated in recent years and corresponding cDNAs have been cloned. This review summarizes the structure and function of mammalian proteins which are essential components of the constitutive splicing machinery.