A panel of 65 systemic lupus erythematosus (SLE) and 61 normal‐derived human hybridoma auto‐antibodies was studied for cytoskeletal reactivity, using an indirect immunofluorescence method. Reactivity with the cytoskeleton was expressed 3 times more frequently in the SLE‐derived antibody group and included intermediate filaments, microfilaments, microtubules, and centrioles. By immunoblot analysis, the antigenic specificity of intermediate filament—reactive SLE hybridoma antibodies was not restricted to vimentin, but included cytokeratins and desmin. The antibodies were also studied for their DNA‐binding, lupus anticoagulant, and rheumatoid factor activities. These autoantibody activities were expressed 3–5 times more frequently in the SLE‐derived group. The ability to bind DNA was not a prerequisite for reactivity with intermediate filament proteins. Our findings suggest that there are at least 2 subsets of cytoskeletal‐reactive hybridoma antibodies: those that recognize epitopes found only on cytoskeletal proteins, and those that recognize epitopes common to both DNA and certain cytoskeletal proteins. In addition, we hypothesize that there may be a third subset of antibodies that recognize a phosphate‐containing moiety (phospholipid or phosphoprotein) associated with cytoskeletal filaments.