We previously showed that the major Zn-binding protein, metallothionein (MT) is a critical target for nitric oxide (NO) with resultant increases in labile Zn. We now show that NO donors also affected the activity of the metal responsive transcription factor MTF-1 that translocates from the cytosol to the nucleus in response to physiologically relevant increases in intracellular Zn and transactivates MT gene expression. Exposing mouse lung endothelial cells (MLEC) to ZnCl(2) or the NO donor, S-Nitroso-N-acetylpenicillamine (SNAP, 200 microM), caused nuclear translocation of a reporter molecule consisting of enhanced green fluorescent protein (EGFP) fused to MTF-1 (pEGFP-MTF-1). In separate experiments, NO donors induced increases in MT protein levels (Western blot). In contrast, NO did not cause nuclear translocation of EGFP-MTF-1 in MLEC from MT knockouts, demonstrating a central role for MT in mediating this response. These data suggest that S-nitrosation of Zn-thiolate clusters in MT and subsequent alterations in Zn homeostasis are participants in intracellular NO signaling pathways affecting gene expression.