The ZRT1 gene encodes the transporter responsible for high affinity zinc uptake in yeast. ZRT1 is transcribed in zinc-limited cells and its transcription is repressed in zinc-replete cells. In this report, we describe a second, post-translational mechanism that regulates ZRT1 activity. In zinc-limited cells, ZRT1 is a stable, N-glycosylated plasma membrane protein. Exposure to high levels of extracellular zinc triggers a rapid loss of ZRT1 uptake activity. Our results demonstrate that this inactivation occurs through zinc-induced endocytosis of the protein and its subsequent degradation in the vacuole. Mutations that inhibit the internalization step of endocytosis also inhibited zinc-induced ZRT1 inactivation and the major vacuolar proteases were required to degrade ZRT1 in response to zinc. Furthermore, immunofluorescence microscopy showed that ZRT1 is localized to the plasma membrane in zinc-limited cells and that the protein is transferred to the vacuole via an endosome-like compartment upon exposure to zinc. ZRT1 inactivation is a relatively specific response to zinc; cadmium and cobalt ions trigger the response but less effectively than zinc. Moreover, zinc does not alter the stability of several other plasma membrane proteins. Therefore, zinc-induced ZRT1 inactivation is a specific regulatory system to shut off zinc uptake activity in cells exposed to high extracellular zinc levels thereby preventing overaccumulation of this potentially toxic metal.