Regional variation of adipose tissue triglyceride breakdown (lipolysis) has been suggested to play a role for the health consequences of some forms of obesity. Thus, in the present study we investigated the regulation of lipolysis in isolated adipocytes obtained from different fat depots in females. Intra-abdominal adipose tissue (omental) and subcutaneous abdominal adipose tissue were obtained from the same individuals undergoing abdominal surgery (n = 9); in addition, adipocytes from the subcutaneous gluteal region (n = 12) and from mammary adipose tissue (n = 5) were investigated. The lipolytic/antilipolytic properties of epinephrine (EPI), insulin, clonidine, and prostaglandin E2 (PGE2) were investigated. The most prominent observation was that EPI had none or only minor lipolytic effect in adipocytes from the subcutaneous regions, but significantly enhanced lipolysis by approximately 500% in omental adipocytes (P less than .001). In the presence of the alpha 2-adrenergic antagonist, yohimbine, EPI had similar stimulatory effects (fourfold to fivefold) in all fat depots. The antilipolytic compounds, insulin and clonidine, had greatly reduced antilipolytic properties in omental adipocytes as compared with subcutaneous adipocytes (P less than .01 and P less than .05, respectively). On the other hand, PGE2 had similar antilipolytic properties in adipocytes from the various depots. In conclusion, we found great regional variation in the regulation of lipolysis. Particularly, EPI was much more lipolytic in omental adipocytes than in subcutaneous adipocytes, mainly due to an enhanced functional alpha 2-receptor activity in subcutaneous adipocytes. These in vitro data suggest that free fatty acids (FFA) are more readily mobilized from omental adipose tissue than from subcutaneous adipose tissue.