Many neurodegenerative disorders are accompanied by chronic glial activation, characterized by abundant production of pro-inflammatory cytokines, such as interleukin-1 (IL-1). IL-1 disrupts Ca²⁺ homeostasis and stimulates astrocyte reactivity. The mechanisms by which IL-1 induces Ca²⁺ dysregulation are not completely defined. Here we examined how acute and chronic (24-48hr) treatment with IL-1 affect Ca²⁺ homeostasis in freshly dissociated and primary cultured mouse cortical astrocytes. Cytosol free Ca²⁺ ([Ca²⁺]_cyt) was measured with Fura-2 by using digital imaging. Acute application of 10 ng/ml IL-1 induced Ca²⁺ mobilization from intracellular stores and activated store- and receptor-operated Ca²⁺ entry (SOCE and ROCE, respectively) in both freshly dissociated and cultured actrocytes. Treatment of cultured astrocytes with IL-1 for 24 and 48 hr elevated resting [Ca²⁺]_cyt, decreased Ca²⁺ store content (associated with SERCA2b down-regulation) and augmented ROCE. Based on evidence that receptor-operated, but not store-operated Ca²⁺ channels (ROCs and SOCs, respectively) are Ba²⁺ permeable, Ba²⁺ entry was used to distinguish ROCs from SOCs. ROCE was activated by the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In the presence of extracellular Ba²⁺, OAG-induced elevations of cytosolic Ba²⁺ (Fura-2 340/380nm ratio) were significantly larger in astrocytes treated with IL-1. These changes in IL-1-treated astrocytes correlate with augmented expression of TRPC6 protein, which likely mediate ROCE. Knockdown of the TRPC6 gene markedly reduced ROCE. The data suggest that IL-1-induced dysregulation of Ca²⁺ homeostasis is the result of enhanced ROCE and TRPC6 expression. The disruption of Ca²⁺ homeostasis appears to be an upstream component in the cascade of IL-1-activated pathways leading to neurodegeneration.