Homing endonucleases are a class of site-specific DNA endonucleases encoded by open reading frames within introns and inteins. They initiate the mobility of their host element by recognizing intronless or inteinless alleles of their host gene and making a double-strand break. The homing endonucleases are notable for their long target sites and a tolerance for sequence polymorphisms in their substrates. The methods used to study homing endonucleases are similar to those used to study protein-DNA interactions in general. However, some variations and specialized techniques are useful in characterizing homing endonucleases and these methods are discussed.