Summary Recent evidence shows that the potent brain stimulatory effects of amphetamine are controlled by the endocannabinoid (eCB) signaling system. Our laboratory established that the enzyme, α/β-hydrolase domain 6 (ABHD6), represent a novel molecular component of the eCB signaling system. Indeed, this post-synaptic enzyme controls the activity-dependent production of 2-arachidonoylglycerol (2-AG, the most abundant eCB in the brain), and as such controls the levels and efficacy of 2-AG at cannabinoid CB1 receptors (CB1R). We recently evaluated the involvement of ABHD6 in the locomotor responses stimulated by amphetamine in mice and found that its pharmacological inhibition and genetic deletion exerts a profound enhancing effect on the acute amphetamine-stimulated locomotor activity through a CB1R-dependent mechanism. In this R21 grant, we propose to identify brain regions involved in the ABHD6-dependent control of psychostimulants using several recently developed tools, including a brain-penetrant selective inhibitor of ABHD6 (KT-182 and MJN193), a Cre-dependent ABHD6 mouse line (ABHD6lox/lox), a specific antibody for ABHD6, and a CRISPR/Cas9 Slc6a3 (DAT) knockout model of hyperactivity. There are two main questions that will be addressed by this proposal. First, where in the brain is the interaction between ABHD6 and amphetamine occurring? Second, does ABHD6 alone, or in combination with low dose amphetamine, result in paradoxical calming response measured in a mouse model of attention deficit hyperactivity disorder (ADHD)-like phenotypes. Our aims are: 1: Identify brain regions involved in the ABHD6-dependent control of psychostimulants. 2: Establish the dose-dependent effects of ABHD6 inhibition on the paradoxical calming effects of psychostimulants in an animal model of hyperactivity. The completion of these studies will provide foundational results on the molecular mechanism by which ABHD6 regulates psychostimulant behavior in mice. A better understanding of this novel molecular interaction should help optimize the therapeutic use of psychostimulants while reducing their addiction and toxicity profile.