Objective: Adiponectin mRNA expression in isolated subcutaneous and omental adipocytes was examined across a wide range of adiposity to determine whether adiponectin synthesis is impaired in these adipose tissue depots in obese humans. Tumor necrosis factor (TNF)␣ and dexamethasone were tested for inhibitory effects on adiponectin release from human adipocytes in vitro. Research Methods and Procedures: Adipocytes were isolated by collagenase digestion of abdominal adipose tissue obtained from subjects undergoing surgical procedures or outpatient needle biopsy. Adiponectin and leptin mRNA were quantitated by real-time reverse transcriptase-polymerase chain reaction. Adiponectin and leptin secretion from isolated adipocytes treated with dexamethasone or TNF␣ were determined by radioimmunoassay. Results: There was a significant negative correlation between adiponectin gene expression and BMI in subcutaneous adipocytes from 32 women (r ϭ 0.420; p ϭ 0.02). Adiponectin mRNA was also significantly correlated with serum adiponectin (r ϭ 0.44; p ϭ 0.03; n ϭ 25). There was no correlation between adiponectin mRNA expression and BMI in omental adipocytes from 29 women. Leptin mRNA was significantly and positively correlated (r ϭ 0.484; p ϭ 0.01) with BMI in the same omental adipocyte mRNA preparations. In subcutaneous adipocytes from lean subjects, TNF␣ inhibited adiponectin release by 7.4 Ϯ 1.2% (n ϭ 9, p Ͻ 0.05) but had no effect on adiponectin release from subcutaneous or omental adipocytes from obese subjects. Dexamethasone significantly inhibited adiponectin release with 24 hours of treatment. Discussion: The data suggest that reduced adiponectin synthesis in subcutaneous adipocytes contributes to lower serum adiponectin levels in obesity and that glucocorticoids regulate adiponectin gene expression in human adipocytes. TNF␣ does not seem to directly inhibit adiponectin synthesis in human adipocytes.