Bowman-Birk inhibitor (BBI) proteins are serine protease inhibitors. First isolated from soybean seeds by Bowman [1] and subsequently characterized by Birk et al. [2], BBIs are found in several plant sources, specially mono-and dicotyledonous seeds [3]. BBIs from dicots usually have a molecular mass of 7-8 kDa and are double-headed serine protease inhibitors, while those from monocots are more variable both in size and inhibitory sites.Like many other cotyledonary proteins, BBIs are the products of a multigene family within the same species [4][5][6] Bowman-Birk serine protease inhibitors are a family of small plant proteins, whose physiological role has not been ascertained as yet, while chemopreventive anticarcinogenic properties have repeatedly been claimed. In this work we present data on the isolation of a lentil (Lens culinaris, L., var. Macrosperma) seed trypsin inhibitor (LCTI) and its functional and structural characterization. LCTI is a 7448 Da double-headed trypsin ⁄ chymotrypsin inhibitor with dissociation constants equal to 0.54 nm and 7.25 nm for the two proteases, respectively. The inhibitor is, however, hydrolysed by trypsin in a few minutes timescale, leading to a dramatic loss of its affinity for the enzyme. This is due to a substantial difference in the k on and k* on values (1.1 lm )1 AEs )1 vs. 0.002 lm )1 AEs )1 ), respectively, for the intact and modified inhibitor. A similar behaviour was not observed with chymotrypsin. The twenty best NMR structures concurrently showed a canonical Bowman-Birk inhibitor (BBI) conformation with two antipodal b-hairpins containing the inhibitory domains. The tertiary structure is stabilized by ion pairs and hydrogen bonds involving the side chain and backbone of Asp10-Asp26-Arg28 and Asp36-Asp52 residues. At physiological pH, the final structure results in an asymmetric distribution of opposite charges with a negative electrostatic potential, centred on the C-terminus, and a highly positive potential, surrounding the antitryptic domain. The segment 53-55 lacks the anchoring capacity found in analogous BBIs, thus rendering the protein susceptible to hydrolysis. The inhibitory properties of LCTI, related to the simultaneous presence of two key amino acids (Gln18 and His54), render the molecule unusual within the natural Bowman-Birk inhibitor family.Abbreviations BApNA, N-benzoyl-DL-arginine-p-nitroanilide; BBI, Bowman-Birk inhibitor; COSY-DQf, two-dimensional correlation spectroscopy doublequantum filtered; C.S.I., chemical shift index; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt; GPpNA, N-glutaryl-L-phenylalanine-pnitroanilide; LCTI, Lens culinaris trypsin inhibitor; LCTI*, Lens culinaris trypsin inhibitor hydrolysed form; MD, molecular dynamics; MSTI, Medicago scutellata trypsin inhibitor; PSTI-IVb, Pisum sativum trypsin inhibitor isoform IVb; SA, simulated annealing; sBBI, soybean Bowman-Birk inhibitor; SFTI, sunflower trypsin inhibitor.