The immunofluorescence technique using HEp-2 cells is widely used for the detection of antinuclear antibodies in sera of patients with connective tissue diseases. Among different patterns of nuclear staining the incidence of peripheral pattern which is mainly produced by anti double-stranded DNA antibodies has been markedly decreased after using HEp-2 cells as nuclear substrates. Thirty-five sera in interphase cells and 44 sera in metaphase cells showed peripheral pattern at 1:1 serum dilution from 70 patients with various connective tissue diseases including 46 systemic lupus erythematosus. Metaphase cells nuclei were more sensitive to peripheral pattern than those in interphase cells. Assuming that the incidence of peripheral pattern at 1:1 dilution was 100%, the incidence of peripheral pattern at 1:20 serum dilution dropped into 57.1% in interphase cells and 38.6% in metaphase cells, respectively. The incidence of peripheral pattern at 1:40 dilution dropped into 65.7% in interphase cells and 56.8% in metaphase cells, respectively. Mean value and incidence of anti double-stranded DNA antibodies detected by RIA were significantly higher in sera with peripheral pattern than in sera without peripheral pattern. However, there were some sera which showed peripheral pattern without anti double-stranded DNA antibodies, or there are a few sera which had anti double-stranded DNA antibodies without peripheral pattern. The drop of the incidence of peripheral pattern might be due to interference effect of multiple different antinuclear antibodies. It is necessary to detect anti double-stranded DNA antibodies directly by radioimmunoassay or enzyme-linked immunosorbant assay if occurrence of anti double-stranded DNA antibodies is clinically suspected without peripheral immunofluorescent pattern.