To gain direct access to the interstitial fluid (ISF), a new technique called open-flow microperfusion has been evaluated. This method is based on a double-lumen catheter with macroscopic (0.3-0.5 mm diameter) perforations that is inserted into the subcutaneous adipose tissue and constantly perfused. Thus partial equilibration between the ISF and the perfusion fluid occurs. The glucose concentration of the ISF was determined by established (zero flow rate, no net flux, and recirculation procedures) and new (ionic reference and suction technique) calibration methods by use of open-flow microperfusion. The data show that 1) the glucose concentration in the ISF is significantly lower than the corresponding arterialized venous plasma values during basal steady-state conditions (adipose tissue 3.2 +/- 0.10 mM, plasma 5.27 +/- 0.12 mM) as well as during hyperglycemic clamp experiments (adipose tissue 7.3 +/- 0.13 mM, plasma 9.91 +/- 0.16 mM), and 2) it is possible to determine the recovery continuously by using the ion concentration of the ISF as an internal standard (ionic reference).