Electrospray ionization mass spectrometry has been applied to the structure assignment of seven new microcystins (1-7), obtained from cultured Anabaena sp. strain 186. The seven new microcystins contain the dehydroalanine (Dha) or L-Ser unit instead of the N-methyldehydroalanine unit and the L-Glu and/or its delta-methyl ester [E(OMe)] units at the two variable L-amino acid units, and the structures were assigned as [Dha7]microcystin-E(OMe)E(OMe) (1), [D-Asp3,Dha7]microcystin-E(OMe)E(OMe) (2), [L-Ser7]microcystin-E(OMe)E(OMe) (3), [D-Asp3,L-Ser7]microcystin-E(OMe)E(OMe) (4), [Dha7]microcystin-EE(OMe) (5), [D-Asp3,Dha7]microcystin-EE(OMe) (6), and [L-Ser7]microcystin-EE(OMe) (7). These microcystins are the first examples containing dicarboxylic amino acids at the two variable L-amino acid units in microcystins.