Mounting evidence from in vitro experiments supports bidirectional interactions between astrocytes and neurons, suggesting glial involvement in neuronal information processing in the brain. Elevation of the cytosolic calcium ion (Ca2+ ) concentration has been suggested to be important for gliotransmission; however, the study of Ca2+ dynamics in cerebral cortical astrocytes in vivo became possible only recently. Here, we describe a set of procedures to monitor Ca2+ concentration fluctuations in a population of astrocytes or in astrocytic processes using in vivo two-photon microscopy. Simultaneous recording of neuronal activity by electrodes and astrocytic activity by imaging is a promising way to reveal the nature of neuron–glia interactions in the brain of living rodents.