OBJECTIVE: To determine the variation in preadipocyte isolation procedure and to assess the number and function of preadipocytes from subcutaneous and omental adipose tissue of obese individuals. RESEARCH METHODS AND PROCEDURES: The preadipocyte number per gram of adipose tissue in the abdominal-subcutaneous and abdominal-omental adipose stores of 27 obese subjects with a BMI of 44 +/- 10 kg/m(2) and an age of 40 +/- 9 years was determined. RESULTS: The assessment of the preadipocyte number was found to be labor intensive and error prone. Our data indicated that the number of stromal vascular cells (SVCs), isolated from the adipose tissue by collagenase digestion, was dependent on the duration of collagenase treatment and the size and the origin of the biopsy. In addition, the fat accumulation and leptin production by differentiated SVCs were dependent on the number of adherent SVCs (aSVCs) in the culture plate and the presence of proteins derived from serum and peroxisome proliferator-activated receptor ligands. DISCUSSION: Using our standardized isolation and differentiation protocol, we found that the number of SVCs, aSVCs, leptin production, and fat accumulation still varied considerably among individuals. Interestingly, within individuals, the number of SVCs, aSVCs, and the leptin production by differentiating aSVCs from both the subcutaneous and the omental fat depots were associated, whereas fat accumulation was not. In obese to severely obese subjects, differences in BMI and age could not explain differences in SVCs, aSVCs, leptin production, and fat accumulation.