Adiponectin, a 30-kDa adipokine hormone, circulates as heavy, medium, and light molecular weight isoforms in mammals. Plasma heavy molecular weight (HMW) adiponectin isoform levels are inversely correlated with the incidence of type 2 diabetes in humans. The objectives of the present study were to characterize adiponectin protein and quantify plasma adiponectin levels in chickens, which are naturally hyperglycemic relative to mammals. Using gel filtration column chromatography and Western blot analysis under nonreducing and non-heat-denaturing native conditions, adiponectin in chicken plasma, and adipose tissue is predominantly a multimeric HMW isoform that is larger than 669 kDa mass. Under reducing conditions and heating to 70-100 C, however, a majority of the multimeric adiponectin in chicken plasma and adipose tissue was reduced to oligomeric and/or monomeric forms. Immunoprecipitation and elution under neutral pH preserved the HMW adiponectin multimer, whereas brief exposure to acidic pH led to dissociation of HMW multimer into multiple oligomers. Mass spectrometric analysis of chicken adiponectin revealed the presence of hydroxyproline and differential glycosylation of hydroxylysine residues in the collagenous domain. An enzyme immunoassay was developed and validated for quantifying plasma adiponectin in chickens. Plasma adiponectin levels were found to be significantly lower in 8- compared with 4-wk-old male chickens and inversely related to abdominal fat pad mass. Collectively, our results provide novel evidence that adiponectin in chicken plasma and tissues is predominantly a HMW multimer, suggesting the presence of unique multimerization and stabilization mechanisms in the chicken that favors preponderance of HMW adiponectin over other oligomers.