Several studies have suggested that the function of glutamate receptor channels can be regulated by protein phosphorylation. Furthermore, a basal level of phosphorylation may be necessary to maintain receptor function. Little is known, however, about the phosphorylation state of glutamate receptor channels in neurons and how it is regulated by synaptic activity. In this study, we have investigated the phosphorylation of the AMPA-preferring glutamate receptor subunit GluR1 in cortical neurons in primary culture. These neurons elaborate extensive processes, form functional synapses, and exhibit spontaneous 4–8 sec bursts of synaptic activity every 15–20 sec. In cultures in which this synaptic activity was suppressed by tetrodotoxin and MK-801, the GluR1 protein was phosphorylated on serine residues within a single tryptic phosphopeptide, as determined by phosphoamino acid analysis and phosphopeptide mapping. This same peptide was basally phosphorylated in recombinant GluR1 receptors transiently expressed in human embryonal kidney 293 cells. Treatment of these synaptically inactive cortical neurons with the adenylyl cyclase activator forskolin resulted in a robust increase in phosphorylation on serine residues on a phosphopeptide distinct from the basally phosphorylated peptide. Again, this same phosphopeptide was observed in recombinant GluR1 receptors isolated from 293 cells coexpressing the catalytic subunit of cAMP- dependent protein kinase. Spontaneous synaptic activity in cultures of cortical neurons resulted in a consistent, rapid (within 10–30 sec) increase in phosphorylation on serine and threonine residues. Interestingly, these phosphopeptides were also phosphorylated when neurons from inactive cultures were stimulated with phorbol esters, which activate protein kinase C. These results indicate that AMPA receptors containing the GluR1 subunit may be regulated by extracellular signals working through the cAMP second messenger system as well as by synaptic activity, possibly acting through protein kinase C. Such regulation by protein phosphorylation may be involved in short- term changes in synaptic efficacy thought to involve the functional modulation of AMPA receptors.