In this contribution, we discuss target site location of restriction endonucleases, which are extremely common among prokaryotes occurring in almost every species. These enzymes specifically recognize and cleave short, often palindromic, sequences on bacteriophage or other kinds of foreign DNA (reviews: Pingoud and Jeltsch 1997, 2001). By cleaving incoming DNA, they protect bacteria against bacteriophage infections acting like an immune system. In addition, restriction endonucleases have an important role in the control of horizontal gene transfer and bacterial evolution (Arber 2000; Jeltsch 2003). The cellular DNA is protected against nucleolytic attack by an accompanying DNA methyltransferase which recognizes the same nucleotide sequence and methylates one adenine or cytosine residue within the site (review: Jeltsch 2002). Since restriction endonucleases and DNA methyltransferases are present at the same time in the cell, there is a kinetic competition between them, which necessitates that the restriction enzyme finds its target site on an invading DNA faster than the methyltransferase. In addition, fast target site location is also important, because the invading bacteriophage DNA has to be degraded before it gains control over the cellular metabolism. Therefore, restriction enzymes were among the earliest examples where facilitated diffusion has been shown to be effective in target site location (Jack et al. 1982; Ehbrecht et al. 1985; Terry et al. 1985).