The placenta plays an important role in the regulation of maternal to fetal transfer of toxic substances, including nonessential metals. Metallothioneins (MTs), which are known to have protective effects against heavy metal toxicity, exist in the placenta, but the exact localization of placental MTs (both MT-I and MT-III) and their physiological role in the placenta exposed to mercury are unclear. The present study was performed to examine the localization of MTs and mercury granules in the placenta of mice exposed to mercury vapor. On gestational day 16, MT-I & II-null and wild-type mice were exposed to mercury vapor at 4.9 to 5.9 mg/m3 for 2 hours. At 24 and 48 hours after exposure, the placentas were examined for mercury distribution (autometallography), MT immunoreactivity, and MT mRNA expression (in situ hybridization). No histological changes were observed in the placentas of either MT-null or wild-type mice. Mercury deposition was demonstrated along the boundary between the junctional zone and the labyrinth zone, as well as in the yolk sac, maternal decidual cells, and labyrinth trophoblasts of both MT-null and wild-type mice. MT-I & -II immunoreactivity, which was confined to wild-type mice, was demonstrated in the yolk sac and decidual cells; mercury was also shown in both structures, suggesting that mercury granules were bound to MTs. MT-III mRNA expression was observed in the yolk sac, decidual cells, and spongiotrophoblasts in both MT-null and wild-type mice. There was, however, no evidence of MT at the boundary between the junctional and labyrinth zones, where substantial mercury deposits were demonstrated. These results suggest that placental MTs and the other unknown molecules may be related to the barrier to the placental transfer of mercury.