We aimed at determining which circulating forms of the adipokine adiponectin that increases lipid oxidation in liver and skeletal muscle are related to ectopic fat in these depots in humans. Plasma total-, high-molecular weight (HMW)-, middle-molecular weight (MMW)-, and low-molecular weight (LMW) adiponectin were quantified by an enzyme-linked immunosorbent assay. Their relationships with liver- and intramyocellular fat, measured using (1)H magnetic resonance spectroscopy, were investigated in 54 whites without type 2 diabetes. Liver fat, adjusted for gender, age, and total body fat, was associated only with HMW adiponectin (r = -0.35, P = 0.012), but not with total-, MMW-, or LMW adiponectin. In addition, subjects with fatty liver (liver fat > or =5.56%, n = 15) had significantly lower HMW- (P = 0.04), but not total-, MMW-, or LMW adiponectin levels, compared to controls (n = 39). Similarly, intramyocellular fat correlated only with HMW (r = -0.32, P = 0.039), but not with the other circulating forms of adiponectin. These data indicate that, among circulating forms of adiponectin, HMW is strongly related to ectopic fat, thus possibly representing the form of adiponectin regulating lipid oxidation in liver and skeletal muscle.