Protein phosphorylation is an important mechanism for the posttranslational modulation of ionotropic glutamate receptors and is subject to regulation by changing synaptic inputs. In this study, we investigated the regulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR1 subunit phosphorylation by cocaine exposure in the rat dorsal striatum in vivo. We found that acute cocaine challenge followed by 6 days of repeated systemic injections of cocaine (20 mg/kg once daily) enhanced the sensitivity of the GluR1 subunit in its phosphorylation at serine 831 (Ser831) in the dorsal striatum. This enhancement of the sensitivity of Ser831 phosphorylation was reduced, at the receptor and ion channel level, by blocking (1) group I metabotropic glutamate receptors (mGluRs), (2) N-methyl-D-aspartate receptors, and (3) L-type voltage-operated Ca(2+) channels. Similar reduction of the enhancement was also induced, at the protein kinase level, by inhibiting (1) protein kinase C, (2) calcium/calmodulin-dependent protein kinases, and (3) c-Jun N-terminal kinases. In addition, inhibition of protein phosphatase 1/2A or calcineurin increased GluR1-Ser831 phosphorylation in the dorsal striatum of normal rats, whereas inhibition of these phosphatases did not further enhance the Ser831 phosphorylation in rats pretreated with 7 daily injections of cocaine. These data suggest that the phosphorylation of AMPA receptor GluR1 subunits at Ser831 is subject to upregulation by acute and repeated cocaine administration. Complex signaling integrations among glutamate receptors, Ca(2+) channels, protein kinases, and protein phosphatases participate in this upregulation.