Metallothionein (MT) has been reported to play important physiological roles in the human body, but the distribution and significance of MT is not fully understood. In order to analyze MT expression, three kinds of polyclonal MT fragment antibodies were developed against NH2-terminal, middle regional and COOH-terminal peptide followed by human MT-IA amino acid sequence in rabbits. The characteristics of these antibodies were studied using competitive immunoassay and immunohistochemical staining. The NH2-terminal antibody (anti-MT-N) had the strongest and clearest immunoreactivity to human and mouse tissues, while COOH-terminal antibody (anti-MT-C) showed species difference because of the 4 amino acid difference in the MT fragment peptide between human and mouse. No reactivity was detected using middle regional residue antibody (anti-MT-M) both on competitive immunoassay and on immunostaining. These results suggest that anti-MT-N is the most applicable antibody to use for immunohistochemistry in human, mouse and other specimens.