Fat distribution varies among individuals with similar body fat content. Innate differences in adipose cell characteristics may contribute because lipid accumulation and lipogenic enzyme activities vary among preadipocytes cultured from different fat depots. We determined expression of the adipogenic transcription factors peroxisome proliferator activated receptor-gamma (PPAR-gamma) and CCAAT/enhancer binding protein-alpha (C/EBP-alpha) and their targets in abdominal subcutaneous, mesenteric, and omental preadipocytes cultured in parallel from obese subjects. Subcutaneous preadipocytes, which had the highest lipid accumulation, glycerol-3-phosphate dehydrogenase (G3PD) activity, and adipocyte fatty acid binding protein (aP2) abundance, had highest PPAR-gamma and C/EBP-alpha expression. Levels were intermediate in mesenteric and lowest in omental preadipocytes. Overexpression of C/EBP-alpha in transfected omental preadipocytes enhanced differentiation. The proportion of differentiated cells in colonies derived from single subcutaneous preadipocytes was higher than in mesenteric or omental clones. Only cells that acquired lipid inclusions exhibited C/EBP-alpha upregulation, irrespective of depot origin. Thus regional variation in adipogenesis depends on differences at the level of transcription factor expression and is a trait conferred on daughter cells.