The use of S1 nuclease to map the start site of a transcription unit is a well-established technique. Based on the method of Berk and Sharp, it has undergone many refinements over the years. S1 nuclease mapping requires a relatively detailed knowledge of the gene structure and sequence data (or a very good restriction map) of the first exon and several hundred bases of upstream sequence. Although S1 nuclease mapping is mainly used to map transcription start sites accurately (to be described in this chapter), this method can also be used to map intron-exon junctions. Note that in order to identify a transcriptional start site unambiguously, S1 nuclease mapping should be used in conjunction with primer extension (Chapter 44).