The aim of the present study was to investigate the role of dopamine D(3) receptors in the rewarding effect and hyperlocomotion induced by a prototypical mu-opioid receptor agonist morphine using dopamine D(3) receptor knock-out mice. The mu-opioid receptor in the brain determined by the [tylosil-3,5-(3)H(N)]-[D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin binding assay was not significantly changed by a deletion of the dopamine D(3) receptor gene. Furthermore, we found that no significant differences in G-protein activation by morphine in the limbic forebrain and lower midbrain were noted between the two genotypes. These results suggest that the function of the mu-opioid receptor itself was not affected by a deletion of the dopamine D(3) receptor gene. To ascertain the morphine-induced rewarding effect in both genotypes, the conditioned place preference paradigm was performed. Deletion of the dopamine D(3) receptor gene resulted in a remarkable enhancement of the morphine-induced rewarding effect. Furthermore, knock-out mice with deletions of the dopamine D(3) receptor revealed a dramatic potentiation of morphine-induced hyperlocomotion. Under these conditions, a loss of the dopamine D(3) receptor gene had no effect on the basal levels of dopamine and the increased dopamine turnover by morphine in the limbic forebrain. These findings provide further evidence that dopamine D(3) receptor contributes to the postsynaptically negative modulation of the mesolimbic dopaminergic pathway that is associated with the rewarding effect and hyperlocomotion through the stimulation of mu-opioid receptors induced by morphine in the mouse.