The specificity of three populations of antibodies to the two metallothionein (MT) isoforms has been characterized using a competitive enzyme-linked immunosorbent assay (ELISA). A polyclonal sheep anti-MT-1 antibody (PAb 1) showed higher affinity to MT-1 than to MT-2 with I50 (concentrations required to achieve 50% inhibition) being 18 and 480 ng for MT-1 and MT-2, respectively. Conversely, a polyclonal rabbit anti-MT-2 antibody (PAb 2) showed higher affinity to MT-2 than to MT-1 (I50 being 250 and 15 ng for MT-1 and MT-2, respectively). A third antibody (PAb 3), isolated by removal of anti-MT-1 antibodies in PAB 2 by immunoaffinity purification, showed no cross-reactivity with MT-1. This result suggests that there are two populations of antibodies which cross-react specifically with the MT isoforms. Rat tissue MT concentrations were quantified by ELISA using the three different antibodies and silver-saturation method. The sum of MT isoform concentrations (ELISA estimation using antibodies PAb 1 and PAb 3) were not significantly different (p < 0.05) from the total MT concentrations obtained by silver-saturation method. Therefore, the ELISA can be used to estimate the relative quantities of the two MT isoforms in rat tissues. The predominant isoform in rat tissues was MT-2 and the proportion of MT-1 increased with induced synthesis of MT by metal injection.