This paper reports an analysis of the protein composition of highly purified mammalian spliceosomes isolated by a two-step large-scale affinity chromatography procedure. Splicing complexes were assembled in vitro on biotinylated pre-mRNA, fractionated by gel filtration, and then affinity-purified by binding to avidin-agarose. The purified spliceosomes are unexpectedly complex, containing at least 50 proteins that range in molecular mass from less than 14 to 200 kDa. Three complexes that assemble in the absence of ATP were also purified and characterized. These include a complex enriched in the small nuclear ribonucleoprotein particle U1 and non-specific complexes assembled either on pre-mRNA or an RNA lacking splice sites. Comparison between these complexes and the spliceosome revealed a distinct set of pre-mRNA-specific proteins and a set of proteins that bind to pre-mRNA only in the presence of ATP. Proteins in these two classes, many of which do not correspond in size to known small nuclear ribonucleoprotein particle proteins, are strong candidates for functional splicing components.