The detailed spectral changes observed in the absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra upon addition of Cd2+ to rat liver Cd,Zn-metallothionein (MT) are reported. Results from dialysis experiments clearly demonstrate that up to 8.6 mole equivalents of Cd2+ can be bound to this protein. The excess Cd2+ ions bound appear to have lower binding constants than those of the first seven Cd2+ ions bound. Red blood cell hemolysate (RBC) can compete with the metallothionein for all Cd2+ bound in excess of seven mole equivalents. Thus the RBC hemolysate method of estimating protein concentrations is shown to be correct when based upon complete loading of all binding sites in MT with Cd2+.