A series of 14 monoclonal antibodies (MAs) has been obtained from a single rat-mouse fusion using gel-excised bovine glial filament (GF) proteins as immunogens. These MAs were characterized by two separate immunochemical assays and by two different immunohistochemical methods. Nine MAs demonstrated specificity for GF proteins. One MA also recognized an epitope shared by intermediate filaments (IF) of the vimentin class (VF). Using the enzyme-linked immunosorbent assay, four of the MAs recognized 200,000, 150,000, and 51,000 dalton proteins, suggesting that these MAs were specific for GF proteins (the 51,000 dalton protein) and neurofilament (NF) proteins (the two high-molecular-weight proteins). However, in both of the immunohistochemical assay systems, these MAs stained neurons and their processes but not astroglial cells. These observations strongly suggest that the 51,000-dalton protein recognized by these four MAs was not derived from GF proteins but instead represents derivatives of NF protein subunits comigrating in gels with GF proteins. These data provide additional information concerning the unique and shared antigenic determinants of the three classes of IF (NF, GF, and VF) of the CNS. In addition, they draw attention to the fact that proteins of certain IF may undergo degradation and comigrate in gels with the proteins of unrelated IF. This emphasizes the need for the use of independent immunochemical and immunohistochemical assays in the characterization of the specificity of MAs.