Objective: To confirm the interaction between cholinergic and opioid systems in alcohol seeking behavior using an animal model. Methods: Experiments were conducted using Conditioned Place Preference(CPP) paradigm. Mice were conditioned with alcohol, nicotine and combination of both. They were then subjected to postconditioning test, in which their preference scores were measured. Following a period of drug abstinence, they were reinstated by morphine at doses of 5, 10, 20 and 40 mg/kg BW to induce relapse. Acetylcholinesterase (AChE) activity measurements were performed at the end of the behavioral tests using Ellman’s method. Results: Priming dose of morphine of10mg/kg, 20mg/kgand40mg/kg BW increased significantly the preference score during relapse to alcohol compared with the score in post conditioning test. AChE activity in animal at the time of relapse was significantly different compared to saline treated group. The highest enzyme activity was shown after priming dose of 20 mg/kg BW in group conditioned with alcohol and nicotine. There was no significant differences between the activity of AChE in groups receiving 5 mg/kg, 10 mg/kg, 20 mg/kg and 40 mg/kg BW of morphine challenge. Conclusion: Result of the present study indicated that morphine challenge in alcohol dependent animals during drug abstinence induces relapse to alcohol dependence. This is accompanied by increased AChE activity suggesting cholinergic-opioid system interaction