Toxin production in a large number of Pyrenophora teres isolates have been investigated. During fungal growth, the pH of the medium decreases from 6.5 to about 3.0. Aspergillomarasmine A is the major toxin excreted into the culture medium. Nonenzymatic acid-catalyzed conversion of aspergillomarasmine A to anhydroaspergillomarasmine A proceeds at low pH and is prevented by repeated titration of the culture medium to pH 6.5. The four possible stereoisomers of N-(2-amino-2-carboxyethyl)aspartic acid have been chemically synthesized and their absolute configuration determined. From circular dichroism and NMR spectroscopy and by measurements of specific optical rotation, the LL-form is identified as the stereoisomer produced by P. teres. Biosynthetic experiments using radioisotopes demonstrate that the LL-isomer of N-(2-amino-2-carboxyethyl) aspartic acid is a direct precursor of aspergillomarasmine A. Consequently, L-configuration is assigned to the two corresponding asymmetric carbon atoms of aspergillomarasmine A. This is in contrast to earlier reports which had indicated D configuration. The phytotoxicity of anhydroaspergillomarasmine A is comparable with that of L-aspartic acid, whereas LL-N-(2-amino-2-carboxyethyl)aspartic acid exerts strong phytotoxicity in the bioassays as shown previously for aspergillomarasmine A. The amount of LL-N-(2-amino-2-carboxyethyl)aspartic acid which accumulates in the P. teres cultures is low, indicating that aspergillomarasmine A is the toxin which plays the major role in the pathological changes associated with the barley net-spot blotch disease.