Recent studies have suggested a functional link between cortical cholinergic output and attentional task demands, whereby acetylcholine (ACh) release is regulated according to the outcome of ongoing behaviour. To explore this hypothesis we measured ACh efflux in the rat medial prefrontal cortex (mPFC) during between-session manipulations of the cognitive demands of an attentional task. Rats were trained to detect visual stimuli in a five-choice serial reaction time task (5-CSRTT) which involves sustained and divided attention. Following habituation to tethering and implantation with a microdialysis probe in the mPFC, rats were tested in the 5-CSRTT for three consecutive days, with different lengths of stimulus duration. During performance of the 5-CSRTT we measured robust, reproducible, task-related increases in ACh release in the mPFC across all sessions. Variations of the stimulus duration from the standard 0.5 s resulted in the predicted behavioural effects (reductions and increases in choice accuracy with 0.25 s and 5 s, respectively), but there was no evidence of either greater changes in ACh release in the more demanding condition or smaller changes in the less demanding condition. By contrast, in the session with 5-s stimulus duration there was a positive correlation between prefrontal cortical ACh efflux and the total number of trials completed. In summary, the present study shows that ACh efflux in the rat mPFC is increased during performance of a 5-CSRTT, but has found no evidence to support a specific relationship between cholinergic cortical output and attentional performance.