Glutamate receptors are the major excitatory receptors in the vertebrate CNS and have been implicated in a number of physiological and pathological processes. Previous work has shown that glutamate receptor function may be modulated by protein kinase A (PKA)-mediated phosphorylation, although the molecular mechanism of this potentiation has remained unclear. We have investigated the phosphorylation of specific amino acid residues in the C-terminal cytoplasmic domain of the rat kainate receptor subtype 6 (GluR6) as a possible mechanism for regulation of receptor function. The C-terminal tail of rat GluR6 can be phosphorylated by PKA on serine residues as demonstrated using [gamma-32P]ATP kinase assays. Whole cell recordings of transiently transfected human embryonic kidney (HEK) 293 cells showed that phosphorylation by PKA potentiates whole cell currents in wildtype GluR6 and that removal of the cytoplasmic C-terminal domain abolishes this potentiation. This suggested that the C-terminal domain may contain residue(s) involved in the PKA-mediated potentiation. Single mutations of each serine residue in the C-terminal domain (S815A, S825A, S828A, and S837A) and a truncation after position 855, which removes all threonines (T856, T864, and T875) from the domain, do not abolish PKA potentiation. However, the S825A/S837A mutation, but no other double mutation, abolishes potentiation. These results demonstrate that phosphorylation of the C-terminal tail of GluR6 by PKA leads to potentiation of whole cell response, and the combination of S825 and S837 in the C-terminal domain is a vital component of the mechanism of GluR6 potentiation by PKA.