Previously, we demonstrated that enhancing cholinergic activity during a working memory (WM) task improves performance and reduces blood flow in the right anterior middle/superior frontal cortex, an area known to be important for WM. The purpose of this study was to evaluate the interaction between WM task demands and cholinergic enhancement on neural responses in the prefrontal cortex. Regional cerebral blood flow (rCBF) was measured using H215O and positron emission tomography, as 10 young healthy volunteers performed a parametrically varied match-to-sample WM for faces task. For each item, a picture of a face was presented, followed by a delay (1, 6, 11, or 16 sec), then by the presentation of two faces. Subjects were instructed to identify which face they previously had seen. For control items, nonsense pictures were presented in the same spatial and temporal manner. All conditions were performed during an intravenous infusion of saline and physostigmine (1 mg/hr). Subjects were blind to the substance being infused. Reaction time increased significantly with WM delay, and physostigmine decreased reaction time across delay conditions. Significant task-related rCBF increases during saline infusion were seen in superior frontal, middle frontal, and inferior frontal regions, and the response magnitudes in the regions increased systematically with task difficulty. In all of these prefrontal regions, physostigmine administration significantly reduced rCBF during task, particularly at longer task delays, so that no correlation between task delay and rCBF was observed. In the ventral visual cortex, physostigmine increased rCBF at longer task delays in medial regions, and decreased rCBF over delay conditions in lateral cortical areas. These results indicate that, during cholinergic potentiation, brain activity in prefrontal regions is not modulated by increases in WM task demands, and lends further support to the hypothesis that cholinergic modulation enhances visual processing, making the task easier to perform, and thus, compensate for the need to recruit prefrontal cortical regions as task demands increase.