The present study revealed the mechanism underlying the nuclear trafficking of metallothionein (MT). Nuclear localization of MT in digitonin-permeabilized BALB 3T3 cells was enhanced in the presence of a cytosolic factor added as a rat red blood cell lysate by oxidation with H2O2 in a dose-dependent manner, but inhibited with excess glutathione. A cytosolic partner was assumed to bind MT and retain it in the cytoplasm, and its oxidation can mobilize MT to the nuclei on cellular oxidation. Pre-treatment of nuclei with H2O2 did not enhance the localization, and MT that had been localized in the nuclei was washed out, indicating that MT is in the nuclei as a result of a higher rate of uptake by the nuclei than the rate of diffusion from the nuclei. Nuclear localization of lysozyme and nuclear localization signal (NLS)-bearing allophycocyanin were not enhanced by the oxidation in the presence of cytosolic factor, suggesting that the nuclear traffic occurring on oxidation is specific to MT. Moreover, when cells were arrested the cell cycle at the S phase, MT was localized in the nuclei in response to coincidental generation of a feeble reactive oxygen species (ROS). These observations suggest that MT comes localized in the nuclei on the sensing of intracellular oxidation, whereby a cytosolic partner specific to MT comes oxidized as a cargo system, MT being localized as a result of enhanced uptake in the nuclei and re-localized in the cytoplasm diffusely. Nuclear MT was proposed to protect the nuclei from the oxidation occurring with progression of the cell cycle. © 2004 Wiley-Liss, Inc.