The dopamine D3 receptor is expressed mostly in the limbic regions of the brain that influence reward-related behaviors, including responses to psychostimulants. However, the lack of ligands with sufficient D3 receptor specificity has hampered our understanding of its possible involvement in mediating psychostimulant actions. To unravel dopamine D3 receptor function and underlying mechanisms in response to psychostimulants, we used a genetic approach and generated D3 receptor gene mutant mice. Our analysis indicates that the D3 mutant mice exhibit enhanced behavioral sensitivity to cocaine, amphetamine and combined injections of D1- and D2-class receptor agonists. By contrast, the combined electrophysiological effects of the same D1- and D2-class agonists on single neurons within the nucleus accumbens were not changed in the D3 mutant mice. We propose that the D3 receptor modulates responses to psychostimulants by inhibiting the cooperative effects of postsynaptic D1- and other D2-class receptors at systems level. The continuous use of the gene targeting approach will provide essential information regarding the molecular and cellular basis of psychostimulant actions and allow the development of new strategies in the prevention and cure of drug abuse.