Oligonucleotide-limited transcription has been used to prepare a series of transcripts which allowed the positions of termination by T7 RNA polymerase to be characterized. The same technique was used to prepare a set of transcripts from a rabbit beta-globin gene that extend in intervals of two nucleotides from the 3' splice site of IVS-1 into the second exon. Splicing efficiency in a HeLa cell nuclear extract decreased with decreasing length of the 3' exon, although both steps of the splicing reaction could still be detected with as few as four nucleotides in this exon. No evidence was found for a lower limit to the length of the 3' exon below which splicing would not take place. With longer substrates, the rate of the second step of splicing was increased substantially.