Adiponectin is an adipocyte-specific protein secreted as trimer, hexamer and high-molecular-weight (HMW) complex. Several reports have indicated that the biologically active form of adiponectin is HMW, and that HMW adiponectin concentration is correlated with insulin sensitivity and metabolic disorders including diabetes better than total adiponectin concentration. We developed a sandwich ELISA using a monoclonal antibody against HMW adiponectin purified from human serum. The specificity of established ELISA to HMW adiponectin was confirmed by analysis of human serum fractions prepared by gel filtration chromatography. After careful purification of HMW adiponectin for the calibration standard, the concentration was determined by the amino acid analysis, protein concentration assay by Lowry's method and theoretical calculation from amino acid sequence of adiponectin. An appropriate calibration curve was obtained by HMW adiponectin standards and an assay range is from 0.2 to 25 ng/mL. Intra- and inter-assay coefficient of variation (CV) was below 3.0% and 5.1%, respectively. The recovery of added HMW adiponectin was from 98.5% to 99.2%. No interference of blood components was confirmed by adding free and conjugated forms of bilirubin, hemoglobin and lipids. Serum is suitable as samples and could be stored at 10 degrees C in the refrigerator for at least 28 days stably. Our HMW adiponectin-specific ELISA system is simple, without any procedure of sample pretreatment, reliable, suitable for routine analysis and highly useful for elucidating the clinical significance of adiponectin.