The effect of the central and peripheral acetylcholinesterase (AChE) inhibitor, physostigmine (PHY), was examined on spatial memory using a water maze, motor activity as well as acoustic startle response (ASR) and prepulse inhibition (PPI) in C57BL/6J mice. PHY was administered intraperitoneally (IP) at doses of 0.0, 0.01, 0.03, 0.1 and 0.3 mg/kg and the mice were tested 30 min after injection. Administration of PHY reduced motor activity in the open field in a dose-dependent fashion, with notable decreases in activity observed at 0.1 and 0.3 mg/kg. The results also showed that animals receiving 0.1 mg/kg spent more total time in the peripheral zone than in the central zone. The water maze data showed impairment of acquisition and performance of the task, accompanied by a reduced swimming time and enhanced thigmotaxis at a dose of 0.1 mg/kg. We also found that the ASR was significantly decreased after 0.03 and 0.1 mg/kg with no change in PPI. These results indicate that central plus peripheral cholinesterase inhibition (ChEI) decreased ASR, which is contrary to our previous experiments with the peripheral ChEI pyridostigmine bromide (PB), suggesting different involvement of cholinergic systems in modulating ASR in mice.