Glial fibrillary acidic (GFA) protein and desmin were purified from bovine brain and large intestine, respectively, and used in a comparison of the major protein components of two classes of intermediate filaments, the glial and smooth muscle filaments. The proteins are similar in size, charge, and amino acid composition, but clearly distinct. By sodium dodecyl sulfate-gel electrophoresis, GFA protein is about 5,000 daltons smaller than desmin. GFA protein is composed of three isoelectric variants which are all slightly more basic than the two variants observed for desmin. One-dimensional peptide mapping following limited proteolysis under denaturing conditions or following cyanogen bromide cleavage demonstrates that the proteins are not closely related in primary structure. Assembly-disassembly experiments reveal that the proteins share solubility properties and that negatively stained preparations of in vitro polymerized filaments are very similar. Limited proteolysis under native conditions demonstrates substructural similarities; comparative peptide mapping following digestion with chymotrypsin and trypsin suggests related core polypeptides of about 37,000 and 21,000 daltons. We conclude that GFA protein and desmin are distinct with respect to primary structure, but probably represent two of the more closely related classes of intermediate filament proteins.