An antiserum has been found in a nonimmunized rabbit which reacts strongly with a system of filaments in various fibroblasts, epithelial cells, macrophages and neuroblastoma. These filaments are distinct from the actin microfilament bundles visualized by an antibody against actin, and they are not affected by brief treatment with cytochalasin B. The pattern of these filaments somewhat resembles that described for microtubules, but the filaments could be clearly distinguished from microtubules by a comparison of their respective immunofluorescent patterns during cell division. In response to the drugs colcemid and vinblastine, the filaments reacting with this preimmune serum condense to form a compact perinuclear coil of fibers, a distribution and behavior in agreement with that previously described for the 10 nm or intermediate filaments studied by electron microscopy. Further evidence supporting our conclusion that this antiserum reacts with intermediate filaments is provided by a comparison of electron micrographs and the immunofluorescent patterns from parallel cell cultures. To identify the antigens reacting with this antiserum we have used the new technique of immuno-autoradiography on SDS gels of whole cell extracts. Two reactive polypeptide chains have been identified with apparent molecular weights of 56,000 and 30,000 daltons.