Besides being activated by G-protein beta/gamma subunits, G-protein activated potassium channels (GIRKs) are regulated by cAMP-dependent protein kinase. Back-phosphorylation experiments have revealed that the GIRK1 subunit is phosphorylated in vivo upon protein kinase A activation in Xenopus oocytes, whereas phosphorylation was eliminated when protein kinase A was blocked. In vitro phosphorylation experiments using truncated versions of GIRK1 revealed that the structural determinant is located within the distant, unique cytosolic C-terminus of GIRK1. Serine 385, serine 401 and threonine 407 were identified to be responsible for the incorporation of radioactive (32)P into the protein. Furthermore, the functional effects of cAMP injections into oocytes on currents produced by GIRK1 homooligomers were significantly reduced when these three amino acids were mutated. The data obtained in the present study provide information about the structural determinants that are responsible for protein kinase A phosphorylation and the regulation of GIRK channels.