Synthetic oligodeoxynucleotides with single methyl phosphonate (mp) substitutions were used for an analysis of the contribution of phosphate contacts to the recognition of the cleavage site by the restriction endonuclease EcoRV. Only in the last position within the recognition sequence, is the methyl phosphonate substitution tolerated by the enzyme. The wild-type enzyme cleaves the Sp diastereomer of the oligodeoxynucleotide GACGATATmpCGTC and the unmodified sequence with equal rates, whereas the Rp diastereomer is cleaved much more slowly. Inspection of the crystal structure of an EcoRV-DNA complex revealed that the non-bridging oxygen atoms of the phosphodiester bond between the T and C bases are in hydrogen bonding distance of the hydroxyl group of the amino acid Thr94. We therefore tried to engineer a variant of EcoRV that would prefer a methyl phosphonate linkage over a normal phosphodiester bond and produced mutants with amino acid exchanges at position 94. One of them, Thr94Val, shows a dramatically reduced activity towards the unmodified DNA and does not accept the Rp diastereomer, but cleaves the Sp diastereomer with the same rate as wild-type EcoRV. Its selectivity, i.e. the ratio of cleavage rates determined for the unmodified and modified substrates, differs by three orders of magnitude from that of the wild-type enzyme.