The mechanisms underlying the postsynaptic localization of neurotransmitter receptors are poorly understood. Recently, the peripheral membrane protein gephyrin has been shown to be essential for the formation of inhibitory glycine receptor clusters in cultured rat spinal cord neurons. In vitro gephyrin binds with high affinity to polymerized tubulin. Here, the interaction of gephyrin with different components of the cytoskeleton was investigated in primary cultures of rat spinal neurons. After treatment with alkaloids affecting the cytoskeleton, the morphology of post-synaptic gephyrin clusters was analyzed by confocal immunofluorescence microscopy. Depolymerization of microtubules by demecolcine reduced both the percentage of cells with postsynaptic gephyrin clusters and the number of clusters/cell. The size of the remaining gephyrin clusters was increased whereas their gephyrin density was significantly lower than under control conditions. Depolymerization of microfilaments by cytochalasin D in contrast generated smaller clusters of increased gephyrin density. Demecolcine also dispersed postsynaptic glycine receptor clusters as revealed by immunostaining with a specific monoclonal antibody. These findings support the view that in vivo gephyrin anchors receptor polypeptides to the cytoskeleton by a complex interaction with microtubules and microfilaments.