Experiments were done to define the stoichiometry of binding of Cu(I) to metallothionein (MT) and to determine its sites of binding in mixed-metal species. Spectrophotometric titrations of rabbit liver Cd7-MT 2, apoMT, and Cd4-alpha-domain with Cu(I) revealed endpoints of 3-4, 4 and 8, and 4 and 6-7 added Cu(I)/mol of MT for the three species respectively. Observed endpoints depended on conditions of the titration and the wavelength chosen for absorbance measurement. Nevertheless, from metal and sulphydryl analyses of titrated proteins that were pretreated with Chelex-100 to remove metal ions from solution, almost all of the cadmium was displaced from Cd7-MT by the addition of 7 Cu(I)/mol of MT. Similarly, 4 Cu(I)/mol of Cd4-alpha-domain completely displaced bound cadmium. The Cu4-alpha-domain was converted into a Cu6-alpha species upon addition of two equivalents of Cu(I)/mol of alpha-domain. Reaction of Cd7-MT with 7, 12 and 20 Cu/mol of MT, followed by reaction with Chelex resin, generated protein samples in each case with about 7 Cu/mol of MT. 111Cd-NMR analysis of the reaction of 111Cd7-MT with Cu(I) showed that nearly co-operative one-for-one replacement of 111Cd occurred and that the beta-domain cluster reacted before the alpha-domain cluster. Two mixed-metal MTs with Cu to Zn ratios approximating 3 to 4 and 6 to 4 were isolated from calf liver. After substitution of Zn with 111Cd, NMR spectra of each protein showed that 111Cd was confined almost completely to the alpha-domain. By inference, about 3 or 6 Cu were bound in the beta-domain of these proteins. Supporting this segregation of metal ions into domains, reaction of Cu6, Zn4-MT with nitrilotriacetate removed zinc exclusively, whereas reaction of Cu6,Cd4-MT with 4,7-phenylsulphonyl-2,9-dimethyl-1,10-phenanthroline extracted only Cu(I). Proteolytic digestion of both products followed by gel filtration demonstrated that Cu(I) and Cd were bound to fragments of the intact protein. Finally, reaction of rabbit liver 111Cd7-MT 2 with Cu10-MT 2 resulted in interprotein metal exchange in which 111Cd-moved from the beta- to the alpha-domain according to NMR analysis. In contrast with the prevalent view that six Cu(I) bind to each domain of MT, the present results show that Cu(I) binds to MT with a minimum stoichiometry of about 7 Cu(I)/mol of MT and can bind to the alpha-domain with stoichiometries of 4 or 6 Cu(I)/mol of MT. Although MTs interacting with 12 or 20 Cu(I)/mol of MT are less stable than that binding about 7 Cu(I)/mol, it appears that MT can bind Cu(I) in multiple stoichiometries.