Objective: Microglia are the main immune- competent cells of the CNS. In vitro data suggest that receptor-mediated Ca²⁺ signals in these cells underlie their activation in response to brain injury and control their executive functions such as release of NO, proinflamatory cytokines and chemokines. Hitherto, however, microglial Ca²⁺ signaling has only been studied in reduced preparations. The aim of our study was to examine microglial Ca²⁺ signaling in vivo. Methods: We analyzed in vivo Ca²⁺ signals in resting cortical microglia combining calcium indicator loading by single-cell electroporation with two-photon imaging. Results: Microglial Ca²⁺ signals were readily evoked by ATP and its analogs but not by other substances known to cause microglial Ca²⁺ transients in vitro. Microglia showed no spontaneous Ca²⁺ transients at rest and only rarely generated Ca²⁺ signals in response to strong neuronal activity. Nonetheless, microglia reliably responded with large, generalized Ca²⁺ transients to a damage of an individual neuron. These responses had a short latency (0.4-4 s) and were localized to the immediate vicinity of the damaged neuron (< 50 mm cell body-to-cell body distance). They were occluded by the application of ATPgS as well as UDP and 2-MeSADP, the agonists of metabotropic P2Y receptors, and they required Ca²⁺ release from the intracellular Ca²⁺ stores. Conclusion: Our in vivo data provide a detailed insight into Ca²⁺ signaling in resting microglia. Furthermore, they identify a novel Ca²⁺ store-operated signal (most likely caused by an activation of metabotropic P2Y receptors), which represents a very sensitive, rapid and highly localized response of microglial cells to brain damage.