We have identified and partially characterized the autoantibodies in sera of 60 patients with chronic fatigue syndrome. Approximately 52% of the sera were found to react with nuclear envelope antigens. The combination of nuclear rim staining observed in immunofluorescence microscopy and immunoblot analysis of highly purified nuclear envelope proteins provided initial characterization of these autoantibodies. Further characterization showed that some sera immunoprecipitated the in vitro transcription and translation product of a human cDNA clone encoding the nuclear envelope protein lamin B1. The autoantibodies were of the IgG isotype. The occurrence of autoantibodies to a conserved intracellular protein like lamin B1 provides new laboratory evidence for an autoimmune component in chronic fatigue syndrome.