The distribution of receptors for a neurotransmitter was investigated cytochemically for the first time in the central nervous system, at synapses established on cells of the ventral horn of the rat cervical spinal cord. Three monoclonal antibodies (mAb's) raised against glycine receptors were used. Immunofluorescent staining already showed discontinuous labeling at the surface of neurons, and immunoenzymatic electron microscopy further revealed that the antigenic determinants were confined to the postsynaptic membrane and concentrated at the level of the synaptic complex. More specifically, one mAb directed against the receptive subunit of the oligomeric receptor recognized an epitope on the extracetlular side of the plasma membrane, whereas two other mAb's bound to the cytoplasmic face. Epitopes for the last two mAb's were more accurately localized with protein A-colloidal gold, using an intermediate rabbit anti-mouse immunoglobulin serum. (a) In addition to the presence of gold particles in areas facing the presynaptic active zone (visualized with ethanolic phosphotungstic acid), the labeling extended beyond this zone for ~50-60 nm, which corresponds to the width of one presynaptic dense projection. (b) The distances between the mid membrane and the gold particles were different for the two mAb's (with means of 21.7 + 8.5 nm and 29.8 + 10.4 nm, respectively). The data suggest that one of the recognized epitopes is close to the plasma membrane, whereas the second protrudes into the cytoplasm. Our results indicate that the receptor is a transmembrane protein which has a restricted spatial distribution on the postsynaptic neuronal surface.Little is known about the organization of receptors for neurotransmitters at central synapses, owing, primarily to the lack of specific high-affinity ligands. In the peripheral nervous system, a-bungarotoxin has been used as a cytochemical marker for acetylcholine receptors localized at the endplate (15, 16). Thus, in the central nervous system, the regional distribution of receptors has only been studied, using agonistic substances, either by light microscopy autoradiography (e.g., for opioid receptors [1 ]) or by electron microscopy, as in the case of opiate (11), benzodiazepine (18), and muscarinic cholinergic (14) receptors. Despite a statistical analysis of the distribution of reduced silver grains, the precise distribution of the labeled molecules with respect to the plasma membrane was difficult to assess due to limitations of the autoradiographic technique. We show in this paper that a direct determination of receptor's distribution, such as that for glycine, is now possible with the help of immunochemistry.In the spinal cord, and several other regions of the central nervous system, glycine is involved in inhibitory synaptic transmission (20); it produces an increase of the postsynaptic chloride conductance (32) and this effect is selectively antagonized by strychnine (20). Recently, the glycine receptor was solubilized in the presence of detergent (22) and aft...