Activation of the cAMP/PKA pathway in the dopaminoceptive neurons of the striatum has been proposed to mediate the actions of various classes of drugs of abuse. Here, we show that, in the mouse nucleus accumbens and dorsal striatum, acute administration of morphine resulted in an increase in the state of phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) at Thr34, without affecting phosphorylation at Thr75. The ability of morphine to stimulate Thr34 phosphorylation was prevented by blockade of dopamine D1 receptors. DARPP-32 knockout mice and T34A DARPP-32 mutant mice displayed a lower hyperlocomotor response to a single injection of morphine than wild-type controls. In contrast, in T75A DARPP-32 mutant mice, morphine-induced psychomotor activation was indistinguishable from that of wild-type littermates. In spite of their reduced response to the acute hyperlocomotor effect of morphine, DARPP-32 knockout mice and T34A DARPP-32 mutant mice were able to develop behavioral sensitization to morphine comparable to that of wild-type controls and to display morphine conditioned place preference. These results demonstrate that dopamine D1 receptor-mediated activation of the cAMP/DARPP-32 cascade in striatal medium spiny neurons is involved in the psychomotor action, but not in the rewarding properties, of morphine.