Oligonucleotides containing 1-(β-D-2'-deoxy-threo-pentofuranosyl)cytosine (dCx) and/or 1-(β-D-2'-deoxy-threo-pentofuranosyl)thymine (dT_x) in place of dC and dT residues in the EcoRII and MvaI recognition site CC^A/_TGG were synthesized in order to investigate specific recognition of the DNA sugar-phosphate backbone by EcoRII and MvaI restriction endonucleases. In 2'-deoxyxylosyl moieties of dC_x and dT_x, 3'-hydroxyl groups were inverted, which perturbs the related individual phosphates. Introduction of a single 2'-deoxyxylosyl moiety into a dC·dG pair resulted in a minor destabilization of double-stranded DNA structure. In the case of a dA·dT pair the effect of a 2'-deoxyxylose incorporation was much more pronounced. Multiple dC_x modifications and their combination with dT_x did not enhance the destabilization effect. Hydrolysis of dC_x-containing DNA duplexes by EcoRII endonuclease was blocked and binding affinity was strongly depended on the location of an altered sugar. A DNA duplex containing a dT_x residue was cleaved by the enzyme, but k_cat/K_M was slightly reduced. In contrast, MvaI endonuclease efficiently cleaved both types of sugar-altered substrate analogs. However it did not cleave conformationally perturbed scissile bonds, when the corresponding unmodified bonds were perfectly hydrolyzed in the same DNA duplexes. Based on these data the possible contributions of individual phosphates in the recognition site to substrate recognition and catalysis by EcoRII were proposed. We observed strikingly non-equivalent inputs for different phosphates with respect to their effect on EcoRII-DNA complex formation.