Lipolysis of adipose tissue triglycerides releases glycerol. Twenty-four volunteers, of whom 6 were obese and 13 were women, received a primed-constant infusion of 2H5-glycerol for 120 min during postabsorptive steady-state conditions. Arterial, abdominal venous, and interstitial (microdialysis) samples were taken, and a four-compartment model was applied to assess subcutaneous abdominal adipose tissue glycerol kinetics. Adipose tissue blood flow was measured using 133Xe washout. Venous glycerol concentrations (median 230 μmol/l [interquartile range 210-268]) were consistently greater than those of arterial blood (69.1 μmol/l [56.5-85.5]), while glycerol isotopic enrichments (tracer-to-tracee ratio) were greater in arterial blood (8.34% [7.44-10.1]) than venous blood (2.34% [1.71-2.69], P < 0.01). Microdialysate glycerol enrichment was 1.44% (1.11-1.79), indicating incomplete permeability of glycerol between capillary blood and interstitium. Calculated interstitial glycerol concentrations were between 270 μmol/l (256-350) and 332 μmol/l (281-371) (examining different boundary conditions). The calculated capillary diffusion capacity (ps) was between 2.21 ml · 100 g tissue-1 · min-1 (1.31-3.13) and 3.09 ml · 100 g tissue-1 · min-1 (1.52-4.90) and correlated inversely with adiposity (Rs ≤ -0.45, P < 0.05). Our results support previous estimates of interstitial glycerol concentration within adipose tissue and reveal capillary diffusion capacity is reduced in obesity.