We designed a strategy to identify splicing factors that act by preventing pre-mRNA transport into the cytoplasm. A yeast synthetic intron was inserted into a lacZ gene so that only the pre-mRNA could be translated to produce beta-galactosidase activity. Deletion of either of the 5' splice junction sequence GUAUGU and the branchpoint sequence UACUAAC resulted in a dramatic increase in pre-mRNA translation, indicating its cytoplasmic localization. In rna6 and rna9 mutant strains assayed at the nonpermissive temperature, splicing inhibition occurred simultaneously with a large increase in pre-mRNA translation. Similarly, a point mutation in U1 snRNA decreased splicing efficiency and increased pre-mRNA translation. From these results, we conclude that early acting factors, probably including U1 snRNA, and the RNA6 and RNA9 gene products, interact in vivo with the 5' splice junction and the branchpoint sequence to commit the pre-mRNA to the splicing pathway, thereby preventing its transport to the cytoplasm.