Abstract The striatum and its dopaminergic input participate in temporal processing and numerous studies provide evidence that interactions between dopamine and acetylcholine are critical for striatal functioning. However, the role of local cholinergic innervation of the striatum in behaviors requiring precise timing has not been specifically investigated. Here, we recorded from presumed striatal cholinergic interneurons, identified as tonically active neurons (TANs), in two male rhesus monkeys performing self-initiated movements after specified learned time intervals have elapsed since a visual cue. We found that 38% of all recorded TANs responded to the cue with a pause in firing and the strength of these responses could be modulated according to the duration of the interval being timed and the accuracy of time estimates. By examining the TAN response to the reward itself and by recording from TANs during a Pavlovian procedure in which no action was required, we found evidence that TAN activity modulation may potentially reflect differences in the animal’s prediction of reward. Thus, besides their well-known role in predicting and detecting rewarding events, TANs may generate signals related to the processing of time. Our findings suggest a role of the local cholinergic circuitry in the representation of time within the striatum.