S1 Nuclease degrades single-stranded DNA and RNA endonucleolytically to yield 5´-phosphoryl-terminated products. Double-stranded nucleic acids (DNA:DNA, DNA:RNA or RNA:RNA) are resistant to degradation except with extremely high concentrations of enzyme. The enzyme is used to remove single-stranded termini from double-stranded DNA, for selective cleavage of single-stranded DNA and for mapping RNA transcripts.