De-novo protein synthesis is required in the development of behavioural sensitization. A prior screening test from our laboratory has implicated heat shock protein 70 (Hsp70) as one of the proteins required in this behavioural plasticity. Thus, this study was designed to extend our understanding of the role of Hsp70 in the development of behavioural sensitization induced by a single morphine exposure in mice. First, by employing transcription inhibitor actinomycin D (AD) and protein synthesis inhibitor cycloheximide (CHX), we identified a protein synthesis-dependent labile phase (within 4 h after the first morphine injection) in the development of behavioural sensitization to a single morphine exposure. Second, Hsp70 protein expression in the nucleus accumbens correlated positively with locomotor responses of sensitized mice and, more importantly, the expression of Hsp70 increased within 1 h after the first morphine injection. Third, AD and CHX both prevented expression of Hsp70 and disrupted the development of the single morphine induced behavioural sensitization, which further implied Hsp70 was highly associated with behavioural sensitization. Finally, the selective Hsp70 inhibitor pifithrin-μ (PES) i.c.v. injected in mice prevented the development of behavioural sensitization and, critically, this inhibitory effect occurred only when PES was given within 1 h after the first morphine injection, which was within the labile phase of the development period. Taken together, we draw the conclusion that Hsp70 is crucially involved in the labile phase of the development of behavioural sensitization induced by a single morphine exposure, probably functioning as a molecular chaperone.