Phomopsin A, C 36H 45CIN 6O 12, the main mycotoxin isolated from cultures of Phomopsis leptostromiformis and the cause of lupinosis disease, is a linear hexapeptide containing 3-hydroxy-L-isoleucine, 3,4-didehydrovaline, N-methyl-3-(3-chloro-4,5-dihydroxyphenyl)serine, E-2,3-didehydroaspartic acid, E-2,3-didehydroisoleucine, and 3,4-didehydro-L-proline. The L-configuration of the indicated amino acids was established by a comparison of the N-trifluoroacetyl n-butylester derivatives of the acid hydrolysis products of phomopsin A with samples prepared from authentic amino acids, using capillary gas chromatography on a chiral stationary phase. The E configuration of the two 2,3-didehydro amino acids is based on the products obtained by catalytic hydrogenation and sodium borohydride reduction of phomopsin A followed by acid hydrolysis (for 2,3-didehydroisoleucine) or by analysis of the coupled 13C n.m.r. spectrum of phomopsin A (for 2,3-didehydroaspartic acid). Evidence is presented which shows that the glycine formed during the acid hydrolysis of phomopsin A is derived from the 3,4-didehydrovaline moiety. The sequence of the amino acids was established by heteronuclear 13C{ 1H} selective population inversion (SPI) experiments and by fast atom bombardment (f.a.b.) mass spectrometry of phomopsin A and its derivatives. An X-ray crystallographic study of phomopsin A confirmed the amino acid sequence and showed that the linear hexapeptide is modified by an ether bridge in place of the 5-hydroxy group of the N-methyl-3-(3-chloro-4,5-dihydroxyphenyl)serine and the hydroxy group of the 3-hydroxyisoleucine units. In addition, the X-ray study specified the absolute configuration of phomopsin A as 22 E, 25 E, 3 R, 4 S, 7 S, 10 S, 11 S, 19 S.