Striatal cholinergic interneurons, also known as tonically active neurons (TANs), acquire a pause in firing during learning of stimulus-reward associations. This pause response to a sensory stimulus emerges after repeated pairing with a reward. The conditioned pause is dependent on dopamine from the substantia nigra, but its underlying cellular mechanism is unknown. Usingin vivointracellular recording, we found that both subthreshold and suprathreshold depolarizations in cholinergic interneurons induced a prolonged after-hyperpolarization (AHP) associated with a pause in their tonic firing. The AHP duration was dependent on the level of depolarization, whether elicited by intracellular current injection or by activation of excitatory inputs from the cortex. High-frequency stimulation of the substantia nigra induced potentiation of the cortically evoked excitation and increased the prolonged AHP after the stimulus. These findings from anesthetized animals suggest that a substantia nigra-induced AHP produces stimulus-associated firing pauses in cholinergic interneurons. This mechanism may underlie the acquisition of the pause response in TANs recorded from behaving animals during learning.