The unit-peptide cadystin, the common component of cadmium-binding peptides Cd-BP1 and Cd-BP2 induced in the fission yeast Schizosaccharomyces pombe, was structurally characterized. Metallothioneins, heavy metal-binding proteins involved in metal detoxification in eukaryotic cells, led to the identification of Cd-BP1 (ca 4,000 Da) and Cd-BP2 (ca 1,800 Da) in S. pombe, which consist of cadmium ions and four or two cadystin molecules, respectively. Previous work showed cadystin comprises 3 Cys, 3 Glu, and 1 Gly (seven amino acids) but resists usual Edman degradation, suggesting an unusual structure. This study determined cadystin's structure as H-γ-Glu-Cys-γ-Glu-Cys-Cys-γ-Glu-Gly-OH, with all Glu and two Cys in L-form and one Cys in D-form. Methods included FAB-MS of aminoethylated cadystin, reduction to dethio-cadystin, dansylation, partial acid hydrolysis, enzymatic digestion (thermolysin, carboxypeptidase P, Staphylococcus aureus V8 protease), and absolute configuration analysis via Hare's method (separating enantiomers using diastereomeric Cu(II) chelates with N,N-di-n-propyl-L-alanine).