This study aims at understanding complex interactions between cortical neurons, glia and blood supply developing during the transition from slow-wave sleep to wakefulness. In spite of essential advances from in vitro and culture preparations, the basic mechanisms of glial interactions with their cellular and ionic environment had remained uninvestigated in vivo. Here we approach this issue by performing simultaneous intracellular recordings of cortical neurons and glia, together with measurements of cerebral blood flow (CBF), extracellular K+ concentrations and local field potentials in both anesthetized (ketamine–xylazine) and naturally behaving cats. Under anesthesia, cortical activation was elicited with electric stimulation of cholinergic nuclei (pedunculopontine tegmental in the brainstem and/or nucleus basalis in the basal forebrain). Iontophoretic application of acetylcholine on the recorded cells was also used. In the vast majority of cases (>80%) glial cells were hyperpolarized during electric stimulation or spontaneous activation. This result was also obtained in all cases where iontophoresis was used or when glutamatergic kainate/quisqualate receptors were blocked with 6-cyano-7-nitroquinoxaline-2,3-dione. The glial hyperpolarization was associated with steady neuronal depolarization, increased CBF, lower extracellular K+ concentration, increased membrane resistance, decreased membrane capacitance and persistent positive DC field potentials. In some cases of cortical activation (<20%), glial cells displayed sustained depolarizing potentials, in parallel with neuronal depolarization, decreased CBF and more negative DC field potentials. The above-mentioned effects of cholinergic activation were blocked by the muscarinic antagonist scopolamine. We propose that the glial response to cholinergic activation results from the balance between the direct hyperpolarizing action of acetylcholine and the depolarizing modulation of glutamate from the neighboring neurons, in addition to the modulation of the interglial communication pathway and/or the ionic traffic across blood vessels.