Four Bowman-Birk inhibitors, named LSI-1/4, were isolated and purified from Lathyrus sativus L. seeds. The purification procedure consisted of two cation-exchange chromatography steps, followed by gel-filtration and RP-HPLC. Mass spectrometry analysis of LSI-1/4 inhibitors yielded relative molecular masses of 7914.41 for LSI-1, 6867.67 for LSI-2, 7341.24 for LSI-3 and 7460.01 for LSI-4. N-terminal sequences (up to 30 residues) of LSI-1/4 inhibitors were identical with the exception of sequence positions 21, 27 and 28 and highly similar to those of other Bowman-Birk inhibitors isolated from Leguminosae plants. Inhibitors LSI-1/4 were active towards trypsin and α-chymotrypsin, with IC(50) values for 12.6 nM of trypsin ranging from 4.9 to 24.3 nM. A lower activity was observed against bovine α-chymotrypsin (IC(50) values ranging from 0.5 to 3.4 μM for 15.0 nM of α-chymotrypsin). Peptide mapping of the LSI-1 sequence showed the presence of an Ala residue in the second reactive site, thus explaining the low anti-chymotrypsin activity of this inhibitor. In addition, LSI-1 was endowed with anti-elastase activity, being able to inhibit human leukocyte elastase.