The association between abdominal deposition of adipose tissue and morbidities accompanying obesity may be related to high rates of free fatty acid release from enlarged intra-abdominal stores. To investigate cellular mechanisms that might contribute to enlargement of intra-abdominal adipocytes, lipolysis, triacylglycerol (TG), and diacylglycerol (DG) synthesis from [14C]glucose was measured in abdominal subcutaneous, omental, and mesenteric adipose tissue from severely obese women and men. Subcutaneous adipose tissue from women showed the highest rates of TG synthesis compared with the intra-abdominal site, or any site in men. isoproterenol stimulated TG synthesis more in intra-abdominal than subcutaneous adipose tissue. In the basal state, intra-abdominal adipose tissue from both men and women showed rates of [14C]DG accumulation approximately 50% total [14C]acylglyceride accumulation, whereas, in subcutaneous adipose tissue, [14C]DG accumulation was approximately 25% of total. Basal lipolysis was lower in intra-abdominal than subcutaneous adipocytes. Stimulation of lipolysis reduced [14C]DG accumulation more in intra-abdominal than subcutaneous adipose tissue. Low rates of acylglyceride synthesis in intra-abdominal adipocytes may prevent accumulation of large intra-abdominal fat stores in women.