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.