Dopamine D₁, dopamine D₂, and adenosine A_2A receptors are highly expressed in striatal medium-sized spiny neurons. We have examined, in vivo, the influence of these receptors on the state of phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). DARPP-32 is a potent endogenous inhibitor of protein phosphatase-1, which plays an obligatory role in dopaminergic transmission. A dose-dependent increase in the state of phosphorylation of DARPP-32 occurred in mouse striatum after systemic administration of the D₂ receptor antagonist eticlopride (0.1–2.0 mg/kg). This effect was abolished in mice in which the gene coding for the adenosine A_2A receptor was disrupted by homologous recombination. A reduction was also observed in mice that had been pretreated with the selective A_2A receptor antagonist SCH 58261 (10 mg/kg). The eticlopride-induced increase in DARPP-32 phosphorylation was also decreased by pretreatment with the D₁ receptor antagonist SCH 23390 (0.125 and 0.25 mg/kg) and completely reversed by combined pretreatment with SCH 23390 (0.25 mg/kg) plus SCH 58261 (10 mg/kg). SCH 23390, but not SCH 58261, abolished the increase in DARPP-32 caused by cocaine (15 mg/kg). The results indicate that, in vivo, the state of phosphorylation of DARPP-32 and, by implication, the activity of protein phosphatase-1 are regulated by tonic activation of D₁, D₂, and A_2A receptors. The results also underscore the fact that the adenosine system plays a role in the generation of responses to dopamine D₂ antagonists in vivo.