Although copper is an essential metal, it is capable of catalyzing the formation of reactive oxygen species that can cause intracellular oxidative damage. We investigated the hypothesis that metal- and oxidative-stress responsive signal transduction pathways mediate the cellular and molecular responses associated with copper exposure. Transient transfection assays using COS-7 cells and mouse metallothionein-I (MT-I) or rat NAD(P)H:oxidoreductase 1-based reporter genes demonstrate that copper activates transcription via metal and antioxidant response elements. Concomitant with copper exposures is a decrease the level of total glutathione, and an increase in oxidized glutathione. Depletion of glutathione, prior to copper exposure, increases metal- and oxidative stress-inducible transcription, and cytotoxicity. Pre-treatment with the reactive oxygen scavengers aspirin or vitamin E provides partial protection against copper toxicity, and reduces inducible transcription. Experiments using signal transduction inhibitors and a MTF-1 null cell line demonstrate that copper-inducible MT-I transcription is regulated by protein kinase C and mitogen-activated protein kinase (MAPK) signaling pathways, and requires MTF-1. The results of these studies indicate that copper activates transcription through both metal- and oxidative stress-responsive signal transduction pathways.