A 32-residue polypeptide fragment, designated alpha I, of rat liver metallothionein obtained by subtilisin digestion was studied by 113Cd NMR. The amino acid composition of the fragment corresponded to residues 30-61 of the metallothionein primary structure, and it contained 3.4 g atoms of Cd2+/mol of alpha I-fragment. Four 113Cd resonances were observed, three of which had identical chemical shifts to those assigned to the four-metal cluster in human liver metallothionein-2 under the same pH and buffer conditions. The 5-ppm chemical shift difference between the remaining resonance assigned to the four-metal cluster in the intact protein can be explained to result from the removal of the NH2-terminal polypeptide fragment containing the three-metal cluster. These results provide unambiguous evidence for the two-domain structure of metallothionein, containing a separate three- and a four-metal cluster.