We developed and characterized 14 polyclonal antibodies against peptides whose sequences were predicted from the type 3 serotonin receptor subunit A (5-HT3R-A) cDNA. One such antiserum, 0165, raised against a peptide corresponding to the large putative intracellular loop, immunoprecipitated in vitro translated 5-HT3R-A protein and recognized both recombinant and neuronal 5-HT3R-A protein by Western blot at a high titer. Furthermore, when antiserum 0165 was used to immunolabel brain sections previously hybridized with a riboprobe specific for 5-HT3R-A transcripts, neuronal co-localization of immunoproduct and transcript was widely found throughout the brain. The study of the distribution of 5-HT3R-A-immunoreactivity in the rat central nervous system with antiserum 0165 revealed intensely immunolabeled neurons in the forebrain (isocortex, olfactory regions, hippocampal formation and amygdala), brainstem (sensory and motor nuclei and nuclei of the reticular formation) and spinal cord (dorsal and ventral horn). At the subcellular level, the 5-HT3R-A was found in endomembranes involved in translation (nuclear envelope and endoplasmic reticulum) and in the dendritic plasma-membrane. The present report is the first description of the 5-HT3R-A immunolocalization in the CNS. The wide distribution of the 5-HT3R-A in the brain and spinal cord based on ligand binding, in situ hybridization and immunolocalization studies support its participation in a large array of central nervous system functions.