New protein synthesis has been implicated as necessary for long-lasting changes in neuronal function. Behavioural sensitization to a single exposure to addictive drugs is a form of neuroplasticity, but little is known about the importance of new protein synthesis in the underlying mechanism. This study was designed to investigate the effects of the transcription inhibitor actinomycin D (AD) and the protein synthesis inhibitor cycloheximide (CHX) on induction of behavioural sensitization to a single morphine exposure in mice. In combination with behavioural experiments, changes in gene and protein expression in the mouse nucleus accumbens (NAc) were analysed by RT-PCR array and Western blot respectively. Behavioural sensitization was evident in mice pretreated only once with morphine at the doses of 20 and 40 mg/kg, but not 5 and 10 mg/kg. Mice pretreated with morphine (20 mg/kg) and challenged with a lower dose (5 mg/kg) after a period of 4-21 d washout showed sensitized locomotion. At the doses that did not affect locomotion in mice, AD or CHX significantly suppressed hyperactivity induced by acute treatment, but not challenge with morphine, and blocked induction of behavioural sensitization to a single morphine exposure in a dose-related manner. The results from RT-PCR array and Western blot indicated that the changes of Hsp70 expression in the NAc of mice were associated with behavioural sensitization induced by a single morphine exposure. Together, these findings suggest that induction of behavioural sensitization to a single morphine exposure requires new protein synthesis, potentially involving Hsp70 expression in the NAc of mice.