This study sought to determine whether the choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) enzymes in the brain were affected in a regionally selective manner by chemical and physical stressors: 1) subacute administration of physostigmine (Phy); 2) exercise; and 3) the combination of these two stressors. ChAT and AChE activities in corpus striatum were significantly decreased due to Phy as well as Phy + exercise. This suggests that corpus striatum is affected by chemical stressors but more so by the combination of chemical and physical stressors. The brainstem is the only region which showed inhibition of ChAT activity due to exercise. Subacute Phy also inhibited brainstem ChAT activity. The hippocampus showed significant decrease in ChAT activity due to Phy + exercise but not due to Phy alone. These results suggest that the brain regions involved with control of motor, autonomic and cognitive functions were affected by subacute Phy and exercise. These data are consistent with the hypothesis that the responsiveness of these brain regions to different stressors is a function of the level of ongoing cholinergic activity and that elevations in ACh levels due to AChE inhibition may have long-term effects on the regulation of ChAT and AChE activities through a negative feedback mechanism.