Higher eukaryotic cells express a family of essential splicing factors with a characteristic RNA-binding domain and serine/arginine-rich (SR) motif. These SR proteins, which include SC35 and SF2/ASF, are conserved from Drosophila to man, are required for early steps of spliceosome assembly and can influence splice-site selections. To address their mechanisms of action, SR proteins were examined for their role in committing pre-messenger RNA to the splicing pathway. I report here that SC35 was sufficient on its own to form a committed complex with human beta-globin pre-mRNA. Examination of other SR proteins and pre-mRNA substrates revealed that single SR proteins committed different pre-mRNAs to splicing with pronounced substrate specificity. These results suggest that splicing of different pre-mRNAs may require distinct sets of SR proteins, and that the commitment by SR proteins may be a critical step at which alternative and tissue-specific splicing is regulated.