This study was designed to investigate the effects of neostigmine methylsulfate oh enzyme activity of acetylcholinesterase (AChE) in rat serum, plasma, muscle and liver were investigated in vivo. Twelve Sprague-Dawley Albino male rats, weighing 150-200 g were housed in two groups, each group containing six rats. A single dose (0.05 mg/kg) of neostigmine methylsulfate was injected intramuscularly to the treatment rats. Control rats were given only the same amount of physiological saline. Blood samples for enzyme activity were obtained by cardiac puncture under ether anesthesia one, three, and six hours after the treatment. The tissues were dissected six hours after the treatment. The present study indicated that neostigmine methylsulfate possessed an inhibitory effect on AChE activity in serum, plasma, muscle and liver with the elapse of time. However, the greatest inhibition was found six hours after the injection.