Alternative splicing generates two variants of the GABA_AR 2 subunit, 2S and 2L, which differ by insertion of the amino acid sequence LLRMFSFK into the large cytoplasmic loop between transmembrane domains 3 and 4. This additional sequence within the GABA_AR 2L subunit contains the potential PKC phosphorylation site Serine 343 (Ser343). Here we intended to determine the capacity of these two splice variants to accumulate at inhibitory synaptic terminals and to co-localise with gephyrin, and to find out whether phosphorylation of Ser343 has any effect on GABA_AR distribution. GFP-tagged large cytoplasmic loops of GABA_AR 2S and 2L (GFP::2S/L) were used as surrogates for full-length receptors to study the function of the individual 2S and 2L peptides in transfected spinal cord neurons (SCNs) and COS-7 cells. It was found that GFP::2L displayed a significantly higher capacity to accumulate at inhibitory synapses than GFP::2S. GABA_AR GFP::2S accumulation at inhibitory postsynaptic sites was suppressed to the extent that GFP::2S assumed a diffuse cytosolic distribution. PKC activation facilitated the postsynaptic clustering of GFP::2L but not of GFP::2S. This required the Ser343 residue since substituting Ala343 for Ser343 produced a diffuse cytosolic localisation pattern, like that of GFP::2S. Furthermore, upon PKC activation DsRed2::2L co-localised with gephyrin in transfected COS-7 cells. These results support the idea that alternative splicing regulates the access of GABA_ARs to inhibitory postsynaptic sites in a Ser343 phosphorylation-regulated way. Alternative splicing generates two variants of the GABA_AR 2 subunit, 2S and 2L, which differ by insertion of the amino acid sequence (LLRMFSFK) into the large cytoplasmic loop between transmembrane domains 3 and 4. This additional sequence within the GABA_AR 2L subunit contains the potential PKC phosphorylation site Serine 343 (Ser343). Here we intended to determine the capacity of these two splice variants to accumulate at inhibitory synaptic terminals and to co-localize with gepyhrin in non-neuronal cells, and to find out whether phosphorylation of Ser343 has any effect on GABA_AR distribution. GFP-tagged large cytoplasmic loops of GABA_AR 2S and 2L (GFP::2S/L) were used as surrogates for full-length receptors to study the function of the individual 2S and 2L peptides in transfected spinal cord neurons (SCNs) and COS-7 cells. It was found that GFP::2L displayed a significantly higher capacity to accumulate at inhibitory synapses than GFP::2S. GABA_AR GFP::2S accumulation at inhibitory postsynaptic sites was suppressed to the extent that GFP::2S assumed a diffuse cytosolic distribution. PKC activation facilitated the postsynaptic clustering of GFP::2L but not of GFP::2S. This required the Ser343 residue since exchange of Ser343 against Ala343 produced a diffuse cytosolic localization pattern, like that of of GFP::2S. Furthermore, upon PKC activation GFP::2L co-localized with gephyrin in transfected COS-7 cells. These results support the idea that alternative splicing regulates the access of GABA_ARs to inhibitory postsynaptic sites in a Ser343 phosphorylation-regulated way.