Above recruitment threshold, ocular motoneurons (Mns) show a firing rate linearly related with eye position. Current hypothesis suggests that synaptic inputs are determinant for establishing the recruitment threshold and firing rate gain in these Mns. We investigated this proposal by studying the cholinergic modulation in oculomotor nucleus Mns by intracellular recordings in rat brain slice preparation. All recorded Mns were silent at their resting membrane potential. Bath application of carbachol (10 µm) produced a depolarization and a sustained firing, which was not silenced upon returning membrane potential to the pre-carbachol value via direct current injection. In response to similar membrane depolarization or equal current steps, carbachol-exposed Mns produced higher firing rate and shorter spike afterhyperpolarization phase (AHP) with lower amplitude. The relationship between injected current and firing rate was linear in control and carbachol-exposed Mns. The slope of these relationships (I-F gain) decreased with carbachol exposure. Bath application of agonist and antagonist of nicotinic and muscarinic acetylcholine receptors, and immunohistochemical studies, support the notion that muscarinic receptors are primarily involved in the above-mentioned responses. We conclude that muscarinic inputs play an important role in determining the recruitment threshold and firing rate gain observed in oculomotor Mns in vivo.