Bovine seminal ribonuclease (BS-RNase), the only dimeric protein among the pancreatic-like ribonucleases, is endowed with special structural features and with biological functions beyond enzymatic activity. In solution, the protein exists as an equilibrium mixture of two forms, with or without exchange (or swapping) of the N-terminal arms. After selective reduction and alkylation of the two intrachain disulfide bridges, the dimeric protein can be transformed into a monomeric derivative that has a ribonuclease activity higher than that of the parent dimeric protein but is devoid of the special biological functions. A detailed investigation of the structural features of this protein in solution, in comparison with those of other monomeric ribonucleases, may help unveil the structural details which induce swapping of the N-terminal arms of BS-RNase. The solution structure of the recombinant monomeric form of BS-RNase, as determined by 3D heteronuclear NMR, shows close similarity with that of bovine pancreatic ribonuclease (RNase A) in all regions characterized by regular elements of secondary structure. However, significant differences are present in the flexible regions, which could account for the different behavior of the two proteins. To characterize in detail these regions, we have measured H/D exchange rate constants, temperature coefficients and heteronuclear NOEs of backbone amides for both RNase A and monomeric BS-RNase. The results indicate a large difference in the backbone flexibility of the hinge peptide segment 16-22 of the two proteins, which could provide the molecular basis to explain the ability of BS-RNase subunits to swap their N-terminal arms.