An exciting topic regarding integrative properties of the nervous system is how transient motor commands or brief sensory stimuli are able to evoke persistent neuronal changes, mainly as a sustained, tonic action potential firing. A persisting firing seems to be necessary for postural maintenance after a previous movement. We have studied in vitro and in vivo the generation of the persistent neuronal activity responsible for eye fixation after spontaneous eye movements. Rat sagittal brainstem slices were used for the intracellular recording of prepositus hypoglossi (PH) neurons and their synaptic activation from nearby paramedian pontine reticular formation (PPRF) neurons. Single electrical pulses applied to the PPRF showed a monosynaptic glutamatergic projection on PH neurons, acting on AMPA-kainate receptors. Train stimulation of the PPRF area evoked a sustained depolarization of PH neurons exceeding (by hundreds of milliseconds) stimulus duration. Both duration and amplitude of this sustained depolarization were linearly related to train frequency. The train-evoked sustained depolarization was the result of interaction between glutamatergic excitatory burst neurons and cholinergic mesopontine reticular fibers projecting onto PH neurons, because it was prevented by slice superfusion with cholinergic antagonists and mimicked by cholinergic agonists. As expected, microinjections of cholinergic antagonists in the PH nucleus of alert behaving cats evoked a gaze-holding deficit consisting of a re-centering drift of the eye after each saccade. These findings suggest that a slow, cholinergic, synaptically triggered event participates in the generation of persistent activity characteristic of PH neurons carrying eye position signals.