Cannabinoid receptors and their endogenous ligands have been implicated in the control of psychostimulant induced reward-related behaviours. Their involvement in behavioural sensitisation produced by psychostimulants has still to be determined. This study investigated the effect of a pharmacological blockade of the CB₁ receptor on amphetamine-induced sensitisation in CD1 mice. Furthermore, tissue levels of the two most prominent endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA) were measured in several forebrain structures to gauge a possible relationship between behavioural sensitisation and endocannabinoid transmission. Mice were pre-treated with the CB₁ antagonist SR141716A (3 mg/kg) 30 min before being injected with amphetamine (4 mg/kg) for 7 consecutive days. On the challenge test day, performed after 3 drug abstinence days, the animals were injected with amphetamine (1 mg/kg) only. On each treatment day, locomotor activity was recorded for 60 min following injection. After termination of the behavioural tests, concentrations of AEA and 2-AG were measured in the hippocampus, striatum and in the nucleus accumbens using isotope dilution liquid chromatography/mass spectrometry. ANOVA followed by Tukey test revealed that amphetamine-pretreated mice showed an increased locomotor response to the challenge injection (mean±SEM distance moved: 412.7±57.3 m) compared with vehicle-pretreated animals (188.8±39.3 m) and that the effect of the amphetamine pre-treatment was significantly potentiated by SR141716A (623.8±92.2 m; p< 0.05); the CB₁ antagonist did not significantly affect locomotion when administered alone (260.0±40.2 m; p> 0.05). Student’s t-tests (2-tailed) showed that the sensitized mice and the animals, which had received a single amphetamine injection on the challenge test day, both had increased AEA levels in the striatum (acute: +204.1±36.5%, p< 0.001; sensitised: +178.4±40.2%, p= 0.001) and decreased AEA concentrations in the nucleus accumbens (acute: -35.9±5.4%, p< 0.001; sensitised: -29.7±5.9%, p= 0.003). In addition, the acute amphetamine challenge enhanced AEA concentrations in hippocampus (+33.7±11.5%, p= 0.031). 2-AG levels were increased in the striatum (+51.8±13.8%, p= 0.038) and decreased in the nucleus accumbens (-30.3±9.6%, p= 0.036) of the sensitised animals only.