As a basis for the determination of the metal-coordination topology and the three-dimensional fold of the polypeptide chain, sequence-specific assignments were obtained for the 1H nuclear magnetic resonance (NMR) spectrum of metallothionein-2 from rat liver. The 1H spin systems of the 20 metal-bound cysteines were identified from comparison of two metal-homogeneous protein preparations obtained by reconstitution of the apometallothionein-2 with 113Cd2+ and 112Cd2+, respectively. The identification of the spin systems for the remaining amino acid residues and sequential assignments were then obtained with two-dimensional 1H-NMR experiments at 500 MHz. The assignments are complete except for two backbone amide protons, which were not observed, and the side-chain hydrogen atoms beyond beta CH2 for all eight lysines. The chemical shifts are presented at pH 7.0 and 25 degrees C.