Rabbit liver metallothionein-2 is shown to form covalent bonds with the anticancer agent melphalan, in support of the hypothesis that covalent sequestration by metallothionein constitutes one mechanism for the cross-resistance acquired by cancer patients to therapeutic alkylating agents. Among 20 cysteines in the 2-domain protein, 89% of the first alkylation reaction occurs with 2 that cochelate a zinc cation in the carboxy domain. Computer-supported docking studies indicate a favorable binding site for melphalan near these cysteine sulfhydryl groups. Although folded metallothionein-2 is resistant to trypsin cleavage, alkylation by 1 mol of melphalan allows cleavage by trypsin between the two globular domains.