Antibodies to double-stranded desoxyribonucleic acid (ds-DNA) and to the polymyositis/scleroderma (PM/Scl) complex are regarded as serological markers for systemic lupus erythematosus (SLE) and PM/Scl overlap syndrome, respectively. In a previous study, serum samples were identified that contained antibodies specific for both ds-DNA and PM/Scl. Fourteen of these sera were available for more detailed investigation including the autoantibody profile as determined by several methods including an addressable laser bead assay, Crithidia luciliae indirect immunofluorescence test (CLIFT) and a PM1-Alpha ELISA. Moreover, 300 samples from connective tissue disease patients and 30 PM/Scl positive samples were screened for anti-dsDNA+/PM/Scl + specimens by CLIFT, dsDNA ELISA, and PM1-Alpha ELISA. We confirmed anti-dsDNA and anti-PM/Scl reactivity in 2/7 samples from the previous study. One sample had also anti-chromatin and anti-SS-A reactivity and the second sample was oligoreactive. In addition, 2/300 (0.7%) unselected samples from connective tissue disease patients were identified with anti-dsDNA and anti-PM/Scl reactivity. In a panel of PM1-Alpha positive samples (n = 30) collected regardless of the diagnosis of the patients, no anti-dsDNA reactivity was found. All anti-dsDNA+/anti-PM/Scl+ patients identified fulfilled sufficient criteria to be classified as definite SLE and also had at least one feature of systemic sclerosis (i.e., sclerodactyly and/or Raynaud's phenomenon). Only 1/4 patients had clinical evidence of dermatomyositis. The combination of anti-dsDNA+/anti-PM/Scl + in patients suffering from connective tissue disease is less frequently found than previously described when newer assays are used. Clinically, anti-dsDNA+/anti-PM/Scl+ patients may define a small subgroup of SLE patients with additional features of systemic sclerosis.