The hepatitis C non-structural protein 5A (NS5A) is a Zn2+-binding phosphoprotein essential for viral replication. Expression of NS5A perturbs intracellular Ca2+ levels by an undefined mechanism, activating transcription factors implicated in the chronic pathogenesis of hepatitis infections. Here, we demonstrate that regulated expression of NS5A enhanced the passive leak of Ca2+ from a subset of the endoplasmic reticulum (ER) Ca2+ stores. This action was not replicated by expression of the amphipathic NH2-membrane anchoring domain of NS5A alone, despite targeting to intracellular membranes. Depletion of the NS5A-targeted ER Ca2+ store was prevented under conditions of ample ATP supply suggesting compensatory Ca2+ ATPase activity, but observed under conditions of ATP insufficiency and in intact cells expressing NS5A. (c) 2008 Elsevier Inc. All rights reserved.