SF2 is a 33 kd protein factor required for 5’splice site cleavage and lariat formation during pre-mRNA splicing in HeLa cell extracts. In addition to its essential role in constitutive splicing, SF2 can strongly influence 5’splice site selection. When pre-mRNAs containing multiple &-competing 5’splice sites are spliced in vitro, high concentrations of purified SF2 promote the use of the 5’splice site closest to the 3’splice site. However, SF2 discriminates properly between authentic and cryptic splice sites. These effects of SF2 on splice site selection may reflect the cellular mechanisms that prevent exon skipping and ensure the accuracy of splicing. In addition, alterations in the concentration or activity of SF2, and of other general splicing factors, may serve to regulate alternative splicing in vivo.