Three fractions (designated Ia, Ib, and II) have been isolated from HeLa cell nuclear extracts that are required for splicing of adenovirus and human beta-globin RNA transcripts in vitro. The incubation of two of the fractions (Ib and II) in the presence of ATP resulted in cleavage of precursor mRNA at the 5' splice site and formation of the intron-exon lariat. Addition of fraction Ia to the combination of Ib and II resulted in the formation of spliced RNA and the intron lariat. When fraction II was incubated with precursor RNA in the presence of ATP and the resulting products were sedimented through sucrose gradients, a 30S complex was detected that contained precursor RNA. The combination of fractions Ib and II resulted in the production of a 55S complex that contained the 5' exon as a prominent RNA species. The combination of fractions I (containing Ia and Ib) and II resulted in the formation of the 55S complex and material sedimenting between 40 S and 20 S, in which the predominant RNA species was spliced RNA.