Pathologic impacts in the brain lead to a widespread activation of microglial cells far beyond the site of injury. Here, we demonstrate that glial Ca2+ waves can trigger responses in microglial cells. We elicited Ca2+ waves in corpus callosum glial cells by electrical stimulation or local adenosine triphosphate (ATP) ejection in acute brain slices. Macroglial cells, but not microglia, were bulk-loaded with Ca2+-sensitive dyes. Using a transgenic animal in which astrocytes were labeled by the enhanced green fluorescence protein (EGFP) allowed us to identify the reacting cell populations: the wave activated a Ca2+ response in both astrocytes and non-astrocytic glial cells and spread over hundreds of micrometers even into the adjacent cortical and ventricular cell layers. Regenerative ATP release and subsequent activation of metabotropic purinergic receptors caused the propagation of the glial Ca2+ wave: the wave was blocked by the purinergic receptor antagonist Reactive Blue 2 and was not affected by the gap junction blocker octanol, but enhanced in Ca2+ free saline. To test whether microglial cells respond to the wave, microglial cells were labeled with a dye-coupled lectin and membrane currents were recorded with the patch-clamp technique. When the wave passed by, a current with the characteristics of a purinergic response was activated. Thus, Ca2+ waves in situ are not restricted to astrocytic cells, but broadly activate different glial cell types.