Recent studies suggest that stimulation of ionotropic receptors regulate kinase activity (Yang et al. 2004; Dajas-Bailador et al. 2002). We previously reported that stimulation of GABAC receptors suppressed neuronal apoptosis through activation of cAMP-dependent protein kinase A (PKA) (Yang et al. 2003). PKA is a broad specificity serine/threonine kinase whose fidelity in signaling is maintained through interactions with A kinase anchoring proteins (AKAPs) (Michel & Scott, 2002). To address the mechanisms underlying such signal transduction from ionotropic GABA_C receptor stimulation to PKA activation, the potential involvement of AKAPs was examined. Specific antibodies raised against GABA_C receptor ρ_1-3 subunits precipitated the appropriate ρ subunit, together with AKAP220, and PKA regulatory and catalytic subunits from rat hippocampal tissue lysates. The GABA_C receptor agonist, cis-4-aminocrotonic acid (CACA), activated PKA, as detected by an increased level of phospho-PKA substrate at about 135 kDa in cultured rat hippocampal neurons. This increase was blocked by the specific GABA_C receptor antagonist, (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA) and the PKA inhibitors, KT5720 and H-89. Antisense oligonucleotides for AKAP220 suppressed the GABA_C-induced increase in phospho-PKA substrate 135 kDa. In contrast to the GABA_C-induced selective phosphorylation, a membrane-permeable cAMP, pCPT-cAMP, caused phosphorylation of PKA substrates at 135 kDa, 100 kDa and 75 kDa. These findings suggest that GABA_C receptor stimulation activates PKA to phosphorylate specific substrate(s) via AKAP220.