Adenosine A 2A receptors (A 2A Rs) are well positioned to influence the maladaptive CNS responses to repeated dopaminergic stimulation in psychostimulant addiction. Expression of A 2A Rs in brain is largely restricted to the nucleus accumbens and striatum, where molecular adaptations mediate chronic effects of psychostimulants such as behavioral sensitization. Using a novel forebrain-specific conditional (Cre/loxP system) knockout of the A 2A R in coordination with classical pharmacological approaches, we investigated the involvement of brain A 2A Rs in amphetamine-induced behavioral sensitization. Tissue-specific, functional disruption of the receptor was confirmed by autoradiography, PCR, and the loss of A 2A antagonist-induced motor stimulation. Daily treatment with amphetamine for 1 week markedly enhanced locomotor responses on day 8 in control mice and the sensitization remained robust after a week of washout. Their conditional knockout littermates however showed no sensitization to amphetamine on day 8 and only a modest sensitization following the washout. Pharmacological blockade of adenosine A 2A Rs also was able to block the development (but not the expression) of sensitization in multiple mouse strains. Thus activation of brain A 2A Rs plays a critical role in developing augmented psychomotor responses to repeated psychostimulant exposure.