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SF2, an activity necessary for 5' splice site cleavage and lariat formation during pre-mRNA splicing in v/tro, has been purified to near homogeneity from HeLa cells. The purest fraction contains only two related polypeptides of 33 kD. This fraction is sufficient to complement an SI00 fraction, which contains the remaining splicing factors, to splice several pre-mRNAs. The optimal amount of SF2 required for efficient splicing depends on the pre-mRNA substrate. SF2 is distinct from the hnRNP A1 and U1 snRNP A polypeptides, which are similar in size. Endogenous hnRNA copurifies with SF2, but this activity does not appear to have an essential RNA component. SF2 appears to be necessary for the assembly or stabilization of the earliest specific prespliceosome complex, although in the absence of other components, it can bind RNA in a nonspecific manner. SF2 copurifies with an activity that promotes the annealing of complementary RNAs. Thus, SF2 may promote specific RNA-RNA interactions between snRNAs and pre-mRNA, between complementary snRNA regions, and/or involving intramolecular pre-mRNA helices. Other purified proteins with RNA annealing activity cannot substitute for SF2 in the splicing reaction. Splicing of individual introns in eukaryotic pre-mRNAs takes place by a sequential two-step cleavage-ligation reaction, which has been reproduced m vitro {for reviews, see Green 1986;Padgett et al. 1986; Krainer and Maniatis 19881. In mammalian cell-free systems premRNA splicing has been shown to require multiple nucleoprotein and protein factors {Kr/imer et al.