We have developed a competitive ELISA using a polyclonal antibody that showed specificity to both metallothionin (MT)-1 and MT-2 isoforms in human and animal specimens. The advantage of this ELISA depends on the characteristics of the polyclonal antibody. The NH2 terminal peptide of MT with acetylated methionine was shown to be the epitope of this antibody. The reactivity of this ELISA system with liver, kidney, and brain extracts worked very well for MT1,2 in wild type mice. Extracts of MT-3 knock-out mice also react well this ELISA, as expected, very low in MT 1,2 but in MT1/2 knock-out mice. Detection limits, the ranges of linearity, and reliability coefficients of MT quantification of the ELISA were suitable for determination of MT. From the preliminary study of normal reference ranges, we found that normal MT levels were between approximately 10-30 ng/ml in human serum. We expect in the future to detect cases with low (MT deficiency) and high serum MT concentrations in patients with various diseases, such as brain/liver disorders, and cancers, using this MT ELISA.