A molecular description of the nerve terminal will be required to understand synaptic function fully. The goals of this study were to contribute toward such a description by characterizing a novel synapse- specific protein. A monoclonal antibody library was screened for antibodies to synaptic proteins. The antibodies were then used to isolate cDNA clones by expression screening. Here we report a detailed characterization of the protein reactive with monoclonal antibody F1– 20. Immunohistochemical and biochemical analyses revealed that the F1– 20 protein is synapse associated. Western blot analyses revealed that the F1–20 protein is a brain-specific polypeptide with an apparent molecular weight on SDS-PAGE of 190,000 Da. Northern blot analyses indicated that probes generated from an F1–20 cDNA clone hybridize to a single brain-specific mRNA of approximately 4.8 kilobases. In situ hybridization experiments demonstrated that F1–20 mRNA expression is neuronal specific. Northern and Western blot analyses indicated that F1– 20 mRNA levels increase abruptly at postnatal day 4 and protein levels increase abruptly at postnatal day 7. This corresponds to a period of active synaptogenesis and synaptic maturation in the mouse CNS. We characterized the neuroanatomical distribution of the F1–20 protein by immunohistochemistry, and of the F1–20 mRNA by in situ hybridization. We found that the F1–20 mRNA and protein are expressed nonuniformly in brain. Variation in the expression of the F1–20 protein is complex and reveals patterns also exhibited by probes directed against other synapse-associated molecules. The highest levels of F1–20 protein are found in the cortically organized regions of the brain. The highest levels of F1–20 mRNA are found in long-distance projection neurons. There is also variation in the expression of F1–20 mRNA between different classes of large output neuron, as well as extensive variation in the expression of F1–20 mRNA between different nuclear groups.