Cholinergic neurons originating from the basal forebrain innervate the entire cortical mantle. Choline-sensitive microelectrodes were used to measure the synaptic release of cortical acetylcholine (ACh) at a subsecond resolution in rats performing a task involving the detection of cues. Cues that were detected, defined behaviorally, evoked transient increases in cholinergic activity (at the scale of seconds) in the medial prefrontal cortex (mPFC), but not in a nonassociational control region (motor cortex). In trials involving missed cues, cholinergic transients were not observed. Cholinergic deafferentation of the mPFC, but not motor cortex, impaired cue detection. Furthermore, decreases and increases in precue cholinergic activity predicted subsequent cue detection or misses, respectively. Finally, cue-evoked cholinergic transients were superimposed over slower (at the timescale of minutes) changes in cholinergic activity. Cortical cholinergic neurotransmission is regulated on multiple timescales to mediate the detection of behaviorally significant cues and to support cognitive performance.