Antinuclear antibodies (ANAs) are clinically important indicators of collagen diseases. As corresponding antigens for ANAs vary considerably, patients with collagen diseases usually demonstrate several ANAs coincidentally, making difficult to detect the full spectrum of ANAs in each patient's serum. To design an efficient system for measuring ANAs, an enzyme-linked immunosorbent assay (ELISA) which adsorbs eight kinds of recombinant or purified antigens in each well of a multiwell plate was used and results were compared to those obtained with conventional assays by the fluorescent antinuclear antibodies (FANA), and double immunodiffusion (DID) methods. The positivity rates of 106 sera from patients with collagen diseases and 286 sera from healthy subjects were 92.5% and 5.5%, respectively. Sixty-one of 65 positive sera (93.8%) in the corresponding ANAs positive sera by DID or other conventional assay methods were positive by ELISA. Anti-SSA/Ro antibody could be detected with higher sensitivity by this assay method than with the FANA and DID method, but the sensitivities for anti-Scl-70 antibody and anti-centromere antibody were lower. Application of this ELISA method for measuring ANAs along with the FANA test may be beneficial for diagnosis of collagen diseases.