Autoantibodies against dense fine speckles 70 (DFS70) are found in 10% of healthy individuals, but only in a tiny population of patients with autoimmune rheumatic disease. The antibody may thus be a marker of autoimmune rheumatic disease negativity. To investigate this possibility, we examined the presence of various disease-marker autoantibodies in anti-DFS70 antibody-positive patients with autoimmune rheumatic disease. Serum samples from 500 patients with various types of autoimmune rheumatic disease were examined for anti-DFS70 antibodies by indirect immunofluorescence and immunoblotting. Various disease-marker autoantibodies were measured by enzyme-linked immunosorbent assay. Twenty-two patients were positive for anti-DFS70 antibodies. Eighteen patients also had disease-marker autoantibodies including anti-double stranded DNA, anti-cardiolipin, anti-SS-A, or other antibodies. In one patient with Sjögren syndrome and two patients with dermatomyositis, no disease-marker antibodies were found; however, one patient with dermatomyositis had a concomitant anti-cytoplasmic antibody. All seven systemic lupus erythematosus patients fulfilled the classification criteria for this disease even if anti-nuclear antibody-positive findings were excluded. One patient with morphea had high-titer anti-single stranded DNA antibody. According to this and previous studies, patients with only anti-DFS70 antibody are rarely diagnosed as having autoimmune rheumatic disease. Recognizing dense fine speckle patterns in anti-nuclear antibodies tests is, thus, very important for analysis of laboratory results in rheumatology clinics.