Zn(2+) plays essential roles in biology, and cells have adopted exquisite mechanisms for regulating steady-state Zn(2+) levels. Although much is known about total Zn(2+) in cells, very little is known about its subcellular distribution. Yet defining the location of Zn(2+) and how it changes with signaling events is essential for elucidating how cells regulate this essential ion. Here we create fluorescent sensors genetically targeted to the endoplasmic reticulum (ER) and Golgi to monitor steady-state Zn(2+) levels as well as flux of Zn(2+) into and out of these organelles. These studies reveal that ER and Golgi contain a concentration of free Zn(2+) that is 100 times lower than the cytosol. Both organelles take up Zn(2+) when cytosolic levels are elevated, suggesting that the ER and Golgi can sequester elevated cytosolic Zn(2+) and thus have the potential to play a role in influencing Zn(2+) toxicity. ER Zn(2+) homeostasis is perturbed by small molecule antagonists of Ca(2+) homeostasis and ER Zn(2+) is released upon elevation of cytosolic Ca(2+) pointing to potential exchange of these two ions across the ER. This study provides direct evidence that Ca(2+) signaling can influence Zn(2+) homeostasis and vice versa, that Zn(2+) dynamics may modulate Ca(2+) signaling.