Serine proteinase inhibitors of the squash family were isolated from bitter gourd (Momordica charantia LINN.) seeds by the conventional purification method. Heat treatment of the extract of the seeds allowed removal of large amounts of protein without loss of trypsin and elastase inhibitory activities. From the supernatants thus obtained, the inhibitors were isolated to homogeneity by ion-exchange chromatography, gel filtration, and reversed phase chromatography. One trypsin inhibitor (Momordica charantia trypsin inhibitor-III; MCTI-III) and three elastase inhibitors (Momordica charantia elastase inhibitor-II, -III, and -IV; MCEI-II, -III, and -IV) were newly isolated in addition to trypsin inhibitors MCTI-I and -II and elastase inhibitor MCEI-I previously reported [Hara, S. et. al. (1989). J. Biochem. 105, 88-92]. The primary structures of the four new inhibitors were determined as follows. [sequence: see text] The dissociation constants, Ki, of MCTI-III complex with bovine beta-trypsin, and of MCEI-II, -III, -IV with porcine elastase were determined to be 1.9 x 10(-7) M, 9.4 x 10(-9) M, 4.0 x 10(-9) M, and 4.7 x 10(-9) M, respectively. Although MCTI-III differed from MCTI-I in only two amino acids, having Gly(3) and Gln(13) in place of Arg(3) and Arg(13), the Ki value of MCTI-III was 20-fold larger than that of MCTI-I. Addition of an amino terminal Glu residue, a dipeptide (Glu-Glu-), and a tripeptide (Glu-Glu-Glu-) to MCEI-I strengthened its elastase inhibitory activity by 200-fold.