The gonadotropin-releasing hormone (GnRH) neurons exhibit a unique pattern of episodic activity to control fertility in all mammals. To enable the measurement of intracellular calcium concentration ([Ca2+]i) in adult GnRH neurons in situ, we generated transgenic mice in which the genetically encodable calcium indicator ratiometric Pericam was expressed by approximately 95% of GnRH neurons. Real-time monitoring of [Ca2+]i within adult male GnRH neurons in the acute brain slice revealed that approximately 70% of GnRH neurons exhibited spontaneous, 10-15 s duration [Ca2+]i transients with a mean frequency of 7 per hour. The remaining 30% of GnRH neurons did not exhibit calcium transients nor did a population of non-GnRH cells located within the lateral septum that express Pericam. Pharmacological studies using antagonists to the inositol-1,4,5-trisphosphate receptor (InsP3R) and several calcium channels, demonstrated that [Ca2+]i transients in GnRH neurons were generated by an InsP3R-dependent store-release mechanism and were independent of plasma membrane ligand- or voltage-gated calcium channels. Interestingly, the abolition of action potential-mediated transmission with tetrodotoxin reduced the number of [Ca2+]i transients in GnRH neurons by 50% (p < 0.05), suggesting a modulatory role for synaptic inputs on [Ca2+]i transient frequency. Using a novel transgenic strategy that enables [Ca2+]i to be examined in a specific neuronal phenotype in situ, we provide evidence for spontaneous [Ca2+]i fluctuations in adult GnRH neurons. This represents the initial description of spontaneous [Ca2+]i transients in mature neurons and shows that they arise from an InsP3R-generating mechanism that is further modulated by synaptic inputs.