Partial digestion with T1 RNAase and chemical modification with kethoxal were used to study stability of two hairpin in the proposed secondary structure of the functionally active MS2 RNA fragment MS2 R(--53 leads to 6), containing the regulatory region of the phage replicase cistron. Analysis of the products obtained after the above treatments showed that T1 RNAase and kethoxal attacked predominantly the guanosine residues in the hairpin b of the MS2 R(--53 leads to 6). This implies that in contrast to the structurally stable hairpin a of the polynucleotide, hairpin b appears to be more labile and may exist under the present experimental conditions in equilibrium with its open form. The data of the competition experiments demonstrated that the kethoxal modified MS2 R(-53 leads to 6) and shorter polynucleotide MS2 R(-53 leads to-11) obtained from MS2 R(-53 leads to 6) after T1 RNAase digestion failed to bind with MS2 coat protein. The relatively unstable hairpin b region in the polynucleotide MS2 R(-53 leads to 6) is suggested to play essential role in the complex formation.