Repetitive exposure to opioids elicits sensitization to its psychomotor stimulating effect and environmental stimuli contribute to this effect. This study first developed a procedure that simultaneously measures conditioned hyperactivity and locomotor sensitization, and then investigated the effects of physostigmine on the development and expression of conditioned hyperactivity and locomotor sensitization in rats. Five groups of rats (10-12 rats each) were conditioned with a conditioned stimulus (CS) for 20 min and then drug or saline paired with CS for 2 h daily for 10 days. Rats were tested 20 min on day 18. On day 25, rats were tested 20 min and subsequently 2 h (immediately after morphine injection). Although the 20 min locomotion was not different among the rats on day 1, rats that received 5 mg/kg morphine during conditioning showed higher locomotion than those received saline or 5 mg/kg morphine in the home cage on day 18 and day 25. Rats received 0.1 mg/kg physostigmine and 5 mg/kg morphine during conditioning showed higher locomotion than those received 5 mg/kg morphine on day 18. On day 25, 0.1 mg/kg physostigmine attenuated the conditioned hyperactivity and expression of morphine locomotor sensitization. In contrast, rats received 0.1 mg/kg physostigmine and 5 mg/kg morphine during conditioning showed higher locomotion during 2 h test period than those received 5 mg/kg morphine. In conclusion, this study established a procedure that simultaneously study conditioned hyperactivity and locomotor sensitization. Physostigmine attenuates the expressions but enhances the development of conditioned hyperactivity and sensitization and the possible mechanisms are discussed.