A competitive double antibody radioimmunoassay (RIA) for rat metallothionein (MT) has been developed that has a detection limit of 100 pg and a range of 100 to 100000 pg. The antibody was raised in rabbits against rat MT-2 but it crossreacts equally with MT-1 and MT-2. However, when the assay is done in the presence of 2-mercaptoethanol the antibody is more specific for MT-2. Zn- and Cd- saturated MTs have similar responses in the assay. Addition of Cu(II) to Zn-MT (more than 6 mol Cu/mol MT) in non-reducing conditions modifies the response of the antibody, probably because of Cu(II) oxidation and later MT polymerization. Standard curves developed in the presence of cytosols from brain cortex, hypothalamus or liver did not differ from the standard curve, indicating the absence of interfering substances in the assay. Furthermore, serial dilutions of those cytosols paralleled the response of the standard curve, indicating that the response of the antibody was specific. For comparison, MT levels in some brain areas measured with the present RIA were compared with those measured with an established RIA. In addition, the expected effect of dexamethasone and stress on liver MT levels was clearly identified by this RIA. The results suggest that the present RIA can be used for quantitation of metallothionein.