Proteolytic activation of macromolecular precursors by selective cleavage at the level of basic amino acids is a general process in the biosynthesis of hormones, receptors and viral proteins [1]. The exact mechanism of enzyme-substrate recognition has not yet been fully understood, since only very few endoproteases have been isolated completely, purified thus for. Neverthless, it has been proposed that those basic residues, operating as signal loci for the proteolytic enzyme machinery, are situated next to β-turns and/or loops and, consequently, that the peptide secondary structure plays a key role in the recognition mechanism. To identify these structural features, that could add important pieces of information in understanding the bioactivation reaction, two prohormone systems have been selected for the present study, namely: pro-ocytocin-neurophysin and pro-somatostatin. A series of peptide analogues reproducing the sequence around the dibasic cleavage sites have been examined by means of spectroscopic (CD, FT-IR, NMR) and computational (EM, MD) techniques. The experimental data are discueeed and correlated to the results of the biological assays. Taken together the results suggest the presence of a family of folded conformations in equilibrium with random ones. The folded conformers are characterized by β-turns around the cleavages site, thus exposing the charged Lys and Arg sidechains on one side of the loop. This finding can be of primary importance for the activity-structure relationship for this class of endoproteases.