A site specific endonuclease from Thermus aquaticus, Taq I, cleaves Simian virus 40 (SV40) DNA at a single site. The cleavage site was localized on the physical map by double digestions, using the previously characterized fragments produced by digestion with Hae II, Hae III, AluI, HhaI, HinfI, or BstI. The Taq I site is located at the position that is 56.5% of the unit length from the Eco RI site.