A nuclear extract from HeLa cells has been separated by DEAE-cellulose chromatography into two fractions, both of which are required for mRNA splicing in vitro. Both fractions are heat labile and sensitive to N-ethylmaleimide. The activity of one of the fractions was abolished by preincubation with micrococcal nuclease, while the other fraction was unaffected by this treatment. This abolition indicates an essential nucleic acid component. Fractions I and II are required for the in vitro splicing of human beta-globin and adenovirus transcripts.