The aims of this study were to 1) compare two methods of determining interstitial glycerol concentration in subcutaneous adipose tissue (AT) and 2) determine whether there are regional differences in interstitial glycerol concentration in subcutaneous AT of nonobese, premenopausal women. Microdialysis probes were inserted under local anesthesia into the abdominal (2 probes) and femoral (1 probe) subcutaneous AT in each subject (n = 5) and perfused with a Ringer solution containing 2.5 mM glucose and glycerol in concentrations ranging from 0 to 900 µM. Microdialysis probe relative recoveries and interstitial glycerol concentrations were determined by the no-net-flux method (NNF) and the internal reference method (IR) with the use of [¹³C]glycerol. Microdialysis probe relative recoveries were 57.4 ± 3.6% by NNF and 61.2 ± 10.1% by IR in femoral AT [P = not significant (NS)] and were 55.2 ± 6.0% by NNF and 66.6 ± 4.2% by IR in abdominal AT (P = NS). The calculated interstitial glycerol concentrations determined by NNF and IR were 236.4 ± 42.7 and 241.1 ± 39.6 µM (P = NS) in femoral AT and 151.4 ± 29.7 and 129.4 ± 18.7 µM in abdominal AT (NNF vs. IR, P = NS; femoral vs. abdominal, P < 0.05). It can be concluded that the interstitial glycerol concentration in the femoral AT of nonobese, premenopausal females is ~240 µM and is higher than in abdominal AT (140 µM). Furthermore, the use of a stable isotope of glycerol as an internal reference is suitable for determining interstitial glycerol concentrations in subcutaneous adipose tissue in humans at rest.