In the striatum, dopamine release is inhibited by activation of dopamine D2 autoreceptors. Changes in dopamine release have been attributed to changes in the synthesis of dopamine, which is regulated via phosphorylation of tyrosine hydroxlase (TH), the rate-limiting enzyme in the synthesis of catecholamines. Here, we have studied the involvement of dopamine D2 receptors in the regulation of TH phosphorylation at distinct seryl residues, using phosphorylation site-specific antibodies and a preparation of rat striatal slices. The D2 receptor agonist, quinpirole, reduced basal TH phosphorylation at Ser40 but not at Ser19 or Ser31. Quinpirole was also able to reduce the increase in Ser40 phosphorylation caused by forskolin, an activator of adenylyl cyclase, without affecting the increase in Ser19 phosphorylation produced by the glutamate receptor agonist, N-methyl-d-aspartate (NMDA). In addition, the dopamine D2 receptor agonist reduced both basal and forskolin-stimulated activity of TH, measured as 3,4-dihydroxyphenylalanine (DOPA) accumulation. Quinpirole decreased phosphorylation of Ser40 induced by okadaic acid, an inhibitor of protein phoshatase 1 and 2A and Ro-20-1724, a phosphodiesterase inhibitor. In contrast, quinpirole did not affect the increase in Ser40 phosphorylation caused by the cAMP analogue, 8-Br-cAMP. These data indicate that, in the striatum, activation of dopamine D2 receptors results in selective inhibition of TH phosphorylation at Ser40 via reduction of the activity of adenylyl cyclase. They also provide a molecular mechanism accounting for the ability of dopamine D2 autoreceptors to inhibit dopamine synthesis and release from nigrostriatal nerve terminals.