Biology of GnRH neurons is critically dependent on extracellular Ca2+ (Ca2+o). We evaluated differences in gene expression patterns with low and high Ca2+o in an immortalized GnRH neuron line, GT1-7 cells. Mouse global oligonucleotide microarray was used to evaluate transcriptional differences among the genes regulated by elevated Ca2+o. Our result identified two interferon-γ (IFNγ)-inducible chemokines, CXCL9 and CXCL10, and a beta chemokine, monocyte chemoattractant protein-3 (MCP-3/CCL7), being up-regulated in GT1-7 cells treated with high Ca2+o (3.0 mM) compared with low Ca2+o (0.5 mM). Up-regulation of these mRNAs by elevated Ca2+o was confirmed by quantitative PCR. Elevated Ca2+o stimulated secretion of CXCL10 and MCP-3 but not CXCL9 in GT1-7 cells, and this effect was mediated by an extracellular calcium-sensing receptor (CaR) as the dominant negative CaR attenuated secretion of CXCL10 and MCP-3. CXCL10 and MCP-3 were localized in mouse GnRH neurons in the preoptic hypothalamus. Suppression of K+ channels (BK channels) with 25 nM charybdotoxin inhibited high-Ca2+o-stimulated CXCL10 release. Accordingly, CaR activation by a specific CaR agonist, NPS-467, resulted in the activation of a Ca2+-activated K+ channel in these cells. CaR-mediated MCP-3 secretion involves the PI3 kinase pathway in GT1-7 cells. MCP-3 stimulated chemotaxis of astrocytes treated with transforming growth factor-β (TGFβ). With TGFβ-treated astrocytes, we next observed that conditioned medium from GT1-7 cells treated with high Ca2+ promoted chemotaxis of astrocytes, and this effect was attenuated by a neutralizing antibody to MCP-3. These results implicate CaR as an important regulator of GnRH neuron function in vivo by stimulating secretion of heretofore unsuspected cytokines, i.e., CXCL10 and MCP-3. © 2007 Wiley-Liss, Inc.