Cyl-1 (1) and Cyl-2 (2) are phytotoxic cyclotetrapeptides isolated from a cultured broth of the phytopathogenic fungus, Cylindrocladium scoparium, containing the unique amino acid Aoe (2-amino-8-oxo-9,10-epoxydecanoic acid). The configurations at the α-position of each four amino acids of Cyl-1 and Cyl-2 have been determined by physicochemical data or enzymatic reactions, but the stereochemistry of the epoxide of Aoe remained uncertain. This paper describes the determination of its stereochemistry by CD spectra. The CD spectra of Cyl-1 and Cyl-2 in trifluoroethanol showed a resemblance to those of chlamydocin, with a negative Cotton effect at 288 nm ascribable to the epoxyketone system of Aoe. To eliminate the contribution of the O-methyl tyrosine residue, Cyl-2 was reduced with sodium borohydride to afford dihydro-Cyl-2 (3), whose CD spectrum confirmed the negligible contribution of the tyrosine residue. CD spectra in chloroform showed consistent results with negligible solvent effects on the conformation of the Aoe side chain and O-methyl tyrosine moiety. From the negative Cotton effect at 288 nm, the absolute configuration of the epoxide of both Cyl-1 and Cyl-2 was determined as S. It is interesting that chlamydocin, HC-toxin, Cyl-1, and Cyl-2 all have the S epoxyketone system, and the biological activities of epimeric mixtures of chlamydocin and HC-toxin were only half as strong as the natural compounds, indicating that the configuration of the epoxy ring is commonly very important for the biological activity of these compounds.