Rationale: By enhancing brain anandamide tone, inhibitors of fatty acid amide hydrolase (FAAH) induce anxiolytic-like effects in rodents and enhance brain serotonergic transmission. Mice lacking the faah gene (FAAH-/-) show higher anandamide levels. However, their emotional phenotype is still debated and their brain serotonergic tone remained unexplored. Objectives and methods: In this study, we tested FAAH-/-mice in the social interaction and the open field tests performed under different lighting conditions (dim and bright) since variations of the experimental context were proposed to explain opposite findings. Moreover, by microdialysis performed under dim light, we analyzed their serotonergic transmission in frontal cortex (FC) and ventral hippocampus (vHIPP). Results: In both light conditions, FAAH-/-mice showed reduced emotionality, compared to wt controls, as suggested by the increased rearing and reduced thigmotaxis displayed in the open field and by the longer time spent in social interaction. Basal serotonergic tone was higher in the FC of mutant mice as compared to control mice, while no difference was observed in the vHIPP. K+-induced depolarization produced similar increases of serotonin in both areas of both genotypes. An acute treatment with the CB1 antagonist rimonabant completely abolished the emotional phenotype of FAAH-/-mice and prevented the K+-stimulated release of serotonin in their FC and vHIPP, without producing any effect in wt mice. Conclusions: Our results support the role of FAAH in the regulation of emotional reactivity and suggest that anandamide-mediated hyperactivation of CB1 is responsible for the emotional phenotype of FAAH-/-mice and for their enhanced serotonergic tone. © 2010 The Author(s).