Restriction endonucleases recognize short DNA sequences and cleave double‐stranded DNA at specific sites within or adjacent to the recognition sequences. Restriction endonuclease cleavage of DNA into discrete fragments is one of the most basic procedures in molecular biology. The first method presented in this unit is the cleavage of a single DNA sample with a single restriction endonuclease. A number of common applications of this technique are also described. These include digesting a given DNA sample with more than one endonuclease, digesting multiple DNA samples with the same endonuclease, and partially digesting DNA such that cleavage only occurs at a subset of the restriction sites. A protocol for methylating specific DNA sequences and protecting them from restriction endonuclease cleavage is also presented. A collection of tables describing restriction endonucleases and their properties (including information about recognition sequences, types of termini produced, buffer conditions, and conditions for thermal inactivation) is given at the end of the unit.