GABA(A) receptors are the major sites of fast synaptic inhibition in the brain, where they are predominantly composed of alpha, beta and gamma2 subunits. A role for direct tyrosine phosphorylation of residues 365 and 367 (Y365/367) within the intracellular domain of the gamma2 subunit has been suggested to be important in modulating GABA(A) receptor function, based on the study of recombinant receptors. To address the relevance of these observations for neuronal GABA(A) receptors we have studied the phosphorylation of the gamma2 subunit in the brain. In adult rat brain the gamma2 subunit is phosphorylated on tyrosine residues, including Y365/367 as defined using a phosphospecific antisera. In cultured cortical neurones, phosphorylation of Y365/367 is highly regulated and was only evident upon inhibition of tyrosine phosphatases. We also establish that the tyrosine kinase Src is capable of specifically interacting with the intracellular domains of receptor beta and gamma2 subunits. This may specifically localise tyrosine kinase activity to GABA(A) receptors, facilitating rapid receptor tyrosine phosphorylation upon kinase activation. Together our results suggests that tyrosine phosphorylation of the gamma2 subunit, possibly by closely associated Src, may be a dynamic mechanism for regulating GABA(A) receptor function in the brain.