GroES is a heptameric partner of tetradecameric molecular chaperone GroEL, which ensures the correct folding and assembly of numerous cellular proteins both in vitro and in vivo. This work demonstrates the results of a study of structural aspects of GroES that affect its interaction with GroEL and reassembly. The effect of limited trypsinolysis of GroES on these processes has been studied. It has been shown that limited trypsinolysis of GroES is only strongly pronounced outside the complex with GroEL and results in the cleavage of the peptide bond between Lys20 and Ser21. The N-terminal fragment (~2 kDa) is retained in the GroES particle, which maintains its heptaoligomeric structure but loses the ability to interact with GroEL and dissociates upon a change in the pH from 7 to 8. Trypsin-nicked GroES cannot reassemble after urea-induced unfolding, while the urea-induced unfolding of intact GroES is fully reversible. The reported results indicate the important role of the N-terminal part of GroES subunit in the assembly of its heptameric structure and the interaction with GroEL.