The optimal way of constructing a family of restriction endonuclease EcoRII substrates has been developed. The substrates are DNA-like duplexes containing regularly repeated native or modified sites of this enzyme as well as those of EcoRI and AluI. Synthesis of substrates was performed by water-soluble carbodiimide-induced polycondensation of two nonanucleotides, d(C-C-T-G-G-A-A-T-Tp) and d(C-C-A-G-G-A-G-C-Tp), as constituents of different complementary complexes. The products of reaction (degree of polymerization, 2-20) were isolated by G-200 gel-filtration. The yield of polymers was about 70%. The main products of reaction were dimers when dephosphorylated nonanucleotides (terminators of polycondensation) were used. The thermal stability of DNA-like duplexes is very high. The structure of the polymers obtained has been confirmed by UV-spectroscopy and by CD data as well as by the results of cleavage by EcoRI and AluI restriction endonucleases.