The sera of 103 patients with connective tissue diseases were studied for the presence of anticytoskeletal antibodies by using an indirect immunofluorescence method. PTK2 cells fixed with paraformaldehyde and digitonin were used as substrate. Antibodies to intermediate filaments were detected in sera of 85.7% of polymyositis/dermatomyositis (PM/DM), 62.8% of systemic sclerosis, 54.5% of rheumatoid arthritis, and 37.5% of systemic lupus erythematosus patients, and in 42.5% of normal sera. High titers of these antibodies, which were IgM, were present in 30% of patients' and 5% of normal sera. Antibodies to microfilaments were present in 11.6% of patients' sera and absent in all control sera. These antibodies were IgM or IgG. The switch from an IgM to an IgG antibody was observed in 1 patient. An IgG antibody to the spindle poles and midbody of mitotic cells was present in the serum of 1 patient with the CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias). Antibodies to intermediate filaments and to microfilaments occur commonly in the connective tissue diseases, particularly in PM/DM, and are not detected with substrates or fixation methods used in routine antinuclear antibody testing.