Diacylglycerol acyltransferase (DGAT) could be a rate limiting step in triglyceride (TG) synthesis as it is the final step in this pathway. As such, between depot differences in DGAT activity could influence regional fat storage. DGAT activity and in vitro rates of direct free fatty acid (FFA) storage were measured in abdominal subcutaneous and omental adipose tissue samples from 12 nonobese (BMI<30 kg/m2) and 23 obese men and women (BMI>30 kg/m2) undergoing elective surgery. DGAT activity was greater in omental than in abdominal subcutaneous adipose tissue from nonobese patients (2.0+/-0.9 vs. 0.9+/-0.3 pmol/min/mg lipid, respectively, P=0.003), but not from obese patients (1.4+/-0.6 vs. 1.7+/-0.7 pmol/min/mg lipid, respectively, P=0.10). DGAT activity per unit adipose weight was negatively correlated with adipocyte size (P<0.01) and positively correlated with direct FFA storage in omental (P<0.001) but not in abdominal subcutaneous fat. Tissue DGAT activity varies as a function of adipocyte size, but this relationship differs between visceral and abdominal subcutaneous fat in obese and nonobese humans. Our results are consistent with the hypothesis that interindividual variations in DGAT activity may be an important regulatory step in visceral adipose tissue FFA uptake/storage.