Type III restriction endonucleases recognize nonsymmetric nucleotide sequences. A necessary condition for DNA cleavage is the presence of two unmethylated recognition sites which are inversely ('head-to-head') oriented in the DNA double strand. A DNA substrate possessing one EcoP1 and one EcoP15 site in the head-to-head configuration could not be cleaved by the individual enzymes, however, it was specifically digested in the simultaneous presence of both enzymes. In agreement with the tracking-collision model for the DNA interaction of type III enzymes cleavage could be abolished by Lac repressor bound between the two sites. We conclude that two different type III enzymes can functionally cooperate in the cleavage of DNA.