Contrary to the classic description of acetylcholine (ACh) as a slowly acting neuromodulator that influences arousal states, results from experiments that employed enzyme-selective microelectrodes for the real-time monitoring of ACh release in the cortex of attentional task-performing rats indicate that cholinergic signals manifesting on multiple timescales (seconds, tens of seconds, and minutes) support, and are necessary for, the mediation of defined cognitive operations. Specifically, in the prefrontal cortex, second-based cholinergic signals support the detection of behaviorally significant cues. In contrast to these prefrontal cholinergic transients, performance-associated cholinergic activity that manifested at lower temporal resolution also was observed elsewhere in the cortex. Although tonic cholinergic signal levels were correlated with the amplitudes of cue-evoked cholinergic transients, and the latter with response latencies, the interrelationships and interactions between the multiple cholinergic signaling modes remains unclear. Hypotheses concerning the afferent circuitry contributing to the regulation of second- versus minute-based cholinergic signals are discussed. The discovery of cholinergic transients and their crucial role in cue detection and attentional performance form the basis for new hypotheses about the nature of cholinergic dysfunction in cognitive disorders and offer new targets for the development of treatments for the cognitive symptoms of neuropsychiatric and neurodegenerative disorders.