According to the crystal structure of Cfr 10I restriction endonuclease the acidic residues D134, E71 and E204 are clustered together and presumably chelate metal ion(s) at the active site. Indeed, investigation of the DNA cleavage properties of substitutional mutants of Cfr 10I D134A, E71Q, E71A and E204Q reveals that D134, E71 and E204 residues are essential for cleavage activity, supporting their active site function. Structural comparison indicates that the D134 residue of Cfr 10I spatially overlaps with aspartate residues D91 and D74, from the invariant active site motifs 90PDX 19EAK and 73PDX 15DIK of Eco RI and Eco RV, respectively. However, structural studies in conjunction with mutational analyses suggest that the sequence motif 133PDX 55KX 13E corresponds to the active site of Cfr 10I, but differs from canonical active site motifs of Eco RI and Eco RV. According to the crystal structure of Cfr 10I the serine S188 residue from the 188SVK sequence motif is a spatial equivalent of the acidic residue from the (E/D)XK-part of the active site motif, which is conserved between Eco RI and Eco RV. Site-directed mutagenesis experiments of Cfr 10I, however, revealed that the S188 was not so important for catalysis while the E204 residue located 2.8 Å away indeed was essential for cleavage, suggesting that the glutamate E204 rather than the S188 residue contributes to the metal binding site in Cfr 10I. In addition, model-building studies suggest that mutual interchange of the E204 and S188 residues should lead only to minor positional differences of the carboxylate residues of glutamate side-chains. The double mutant S188E/E204S was therefore prepared by site-directed mutagenesis where the active site motif 133PDX 55KX 13E of Cfr 10I was changed to a canonical motif 133PDX 53EVK, which is similar to that of Eco RI and Eco RV. Interestingly, the double mutant S188E/E204S of Cfr 10I with redesigned active site structure, exhibited 10% of Wt cleavage activity in a λ DNA cleavage assay. Thus, structure guided redesign of the catalytic/metal binding site of Cfr 10I, provides novel experimental evidence to suggest that spatial rather than sequence conservation plays the dominant role in the formation of restriction enzyme active sites.