The strength of synaptic transmission is controlled by the number and activity of neurotransmitter receptors. Yet, little is known about absolute numbers and densities of receptor and scaffold proteins and the stoichiometry of molecular interactions at synapses. Here, we conducted threedimensional and quantitative nanoscopic imaging based on single molecule detections to characterise the ultra-structure of inhibitory synapses and to count scaffold proteins and receptor binding sites. We observed a close correspondence between the spatial organisation of gephyrin scaffolds and glycine receptors at spinal cord synapses. Endogenous gephyrin was clustered at densities of 5000-10000 molecules per µm 2. The stoichiometry between gephyrin molecules and receptor binding sites was approximately 1:1, consistent with a two-dimensional scaffold in which all gephyrin molecules can contribute to receptor binding. The competition of glycine and GABA A receptor complexes for synaptic binding sites highlights the potential of single molecule imaging to quantify synaptic plasticity on the nanoscopic scale.