Although the role of Ca²⁺ in liver transplantation injury has been the object of several studies, direct evidence for alterations in intracellular Ca²⁺ homeostasis after cold preservation-warm reoxygenation (CP/WR) has never been presented. We thus investigated the effects of CP/WR on steady-state Ca²⁺ and responses to a Ca²⁺-mobilizing agonist. Isolated rat hepatocytes were suspended in University of Wisconsin solution, stored at 4°C for 0, 24, and 48 h, and reoxygenated at 37°C for 1 h. Cytosolic Ca²⁺ was measured in single cells by digitized fluorescence videomicroscopy. CP/WR caused a significant increase in steady-state cytosolic Ca²⁺, which was inversely proportional to cell viability. Pretreatment of hepatocytes with an agent that protects mitochondrial function attenuated the increase in steady-state cytosolic Ca²⁺ and improved hepatocyte viability. Ca²⁺ responses to the purinergic agonist ATP also increased significantly as a function of cold storage time. This increase was related to an increase in the size of inositol 1,4,5-trisphosphate-sensitive Ca²⁺ stores and subsequent capacitative Ca²⁺ entry. Thus CP/WR significantly perturbs steady-state hepatocellular Ca²⁺ and responses to Ca²⁺-mobilizing agonists, which may contribute to hepatocyte metabolic dysfunction observed after CP/WR.