The meninges, which envelop and protect the brain, host a dense network of resident macrophages with diverse roles in regulating homeostasis and neuroinflammation. Despite their importance, we have a limited understanding of their behavior in vivo. Many dynamic cellular functions of macrophages involve intracellular Ca 2+ signaling. However, virtually nothing is known about the spatiotemporal Ca 2+ dynamics of meningeal macrophages in vivo. We developed a chronic intravital two-photon imaging approach and related computational analysis tools to interrogate meningeal macrophage Ca 2+ dynamics, at subcellular resolution, in a novel Pf4-Cre:Ai162 conditional GCaMP6s reporter mouse model. Using imaging in awake mice, we characterized Ca 2+ activity in meningeal macrophages at steady state and in response to cortical spreading depolarization (CSD), an aberrant pro-inflammatory brain hyperexcitability event implicated in migraine, traumatic brain injury, and stroke. In homeostatic meninges,