FokI restriction endonuclease recognizes the nonpalindromic pentadeoxyribonucleotide, 5'-GGATG-3':5'-CATCC-3' in duplex DNA and cleaves 9 and 13 nucleotides away from the recognition site. We have reported the presence of two distinct and separable protein domains within this enzyme: one for the sequence-specific recognition of DNA (the DNA binding domain) and the other for the endonucleases activity (the cleavage domain). Our studies have suggested that the two domains are connected by a linker region, which appears to be amenable for repositioning of the DNA-sequence recognition domain with respect to the catalytic domain. Here, we report the construction of several insertion (4-, 8-, 12-, 18-, 19-, or 23-amino acid residues) and deletion (4- or 7-amino acid residues) mutants of the linker region of FokI endonuclease. The mutant enzymes were purified, and their cleavage properties were characterized. The mutants have the same DNA sequence specificity as the wild-type enzyme. However, compared with the wild-type enzyme, the insertion mutants cleave predominantly one nucleotide further away from the recognition site on both strands of the DNA substrate. The four-codon deletion mutant shows relaxed specificity at the cut site while the seven-codon deletion appears to inactivate the enzyme. The DNA binding and cleavage domains of FokI appear to be linked by a relatively malleable linker. No simple linear relationship exists between the linker length and the distance of the cut site from the recognition site. Furthermore, the four-codon insertion mutants cleave DNA substrates containing hemi-methylated FokI sites; they do not cleave fully methylated substrates. These results are best explained as a consequence of protein-protein interactions between the domains.