Excess intra-abdominal fat is associated with a higher risk for type 2 diabetes mellitus and cardiovascular disease, yet little is known about what influences regional adipose tissue accumulation. Adipocytes arise from specialized fibroblast-like preadipocytes within the adipose tissue stromal-vascular compartment. The aim of our study was to determine if there are variations in preadipocyte differentiation between abdominal subcutaneous (SC) and omental (OM) preadipocytes. Abdominal SC and OM preadipocytes were isolated from adipose tissue obtained from 18 subjects (7 men, 11 women), undergoing elective abdominal surgery, by collagenase treatment and filtration/centrifugation. Preadipocytes were placed in culture and then differentiated for 3 weeks in a serum-free medium containing insulin, dexamethasone, isobutylmethylxanthine, and carbaprostacyclin. The cells were then harvested for measurement of cytosolic glycerol phosphate dehydrogenase (GPDH), a marker of terminal differentiation. Data are expressed as a differentiation index (DI), which was the log of the SC/OM ratio of GPDH values for each patient (calculated as 0 for an equivalent SC v OM responses). The mean DI for the group (n = 18) was 0.04, with a 95% confidence interval (CI) of -0.11 to 0.20. The mean DI for men was 0.07 (95% CI, -0.06 to 0.19), and that for women was 0.03 (95% CI, -0.21 to 0.27). This indicates that SC versus OM preadipocyte differentiation responses were not significantly different from each other, either for the group as a whole or when divided by gender. Overall, 8 subjects had a DI favoring SC preadipocyte differentiation, compared to 11 subjects with a DI reflecting greater OM preadipocyte differentiation. There was no correlation of the DI with body mass index or age. Our results indicate that preadipocytes from the abdominal SC adipose tissue depot do not uniformly differentiate more than those from the OM depot.