High-molecular-weight forms of clathrin light chains LCa and LCb contain inserted sequences and are expressed in brain tissue but have not been observed in peripheral tissues. Monoclonal antibodies specific for the high-molecular-weight form of LCb and all forms of LCa were used to analyze their expression in different species and different neuronal cell types. High-molecular-weight light chains were found in bovine, rat, mouse, chicken, and human brain, indicating a conserved pattern of expression. Neuron-specific expression of the high-molecular-weight light chains was suggested by analysis of human brain gray matter and white matter. The former contained a higher proportion of light chains with insertion sequences. Immunohistochemical analysis localized the high-molecular-weight form of LCb to synapses and neuronal perikarya, but not to glial cells. Immunofluorescent labeling of cultured chicken dorsal root ganglia confirmed expression in neurons but not Schwann cells. These results indicate that the high-molecular-weight forms of clathrin light chains are restricted in expression and found in neuronal cells.