Alternative splicing generates two variants of the GABAAR gamma2-subunit, gamma2S and gamma2L, 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 GABAAR gamma2L-subunit contains the potential protein kinase C (PKC) phosphorylation site serine 343 (Ser343). In the present study we intended to determine the capacity of these two splice variants to accumulate at inhibitory synaptic terminals and to colocalize with gephyrin, and to find out whether phosphorylation of Ser343 has any effect on GABAAR distribution. Green fluorescent protein (GFP)-tagged large cytoplasmic loops of GABAAR gamma2S and gamma2L (GFP::gamma2S/L) were used as surrogates for full-length receptors to study the function of the individual gamma2S and gamma2L peptides in transfected spinal cord neurones (SCNs) and COS-7 cells. It was found that GFP::gamma2L displayed a significantly higher capacity to accumulate at inhibitory synapses than GFP::gamma2S. GABAAR GFP::gamma2S accumulation at inhibitory postsynaptic sites was suppressed to the extent that GFP::gamma2S assumed a diffuse cytosolic distribution. PKC activation facilitated the postsynaptic clustering of GFP::gamma2L but not of GFP::gamma2S. This required the Ser343 residue, since substituting Ala343 for Ser343 produced a diffuse cytosolic localization pattern, like that of GFP::gamma2S. Furthermore, upon PKC activation Discosoma Red2-tagged GABAAR gamma2L (DsRed 2::gamma2L) colocalized with gephyrin in transfected COS-7 cells. These results support the idea that alternative splicing regulates the access of GABAARs to inhibitory postsynaptic sites in a Ser343 phosphorylation-regulated way.