The stable isotope-labeled [1,1,2,3,3-H-2(5)]glycerol analyzed by negative-ion chemical ionization (NCI) mass spectrometry has been proven a valid tracer for studying glycerol kinetics in humans. Because of its high technical complexity, NCI mass spectrometry is available to only a few laboratories. Thus, the aim of our study was to create an alternative method for measuring [1,1,2,3,3-H-2(5)]glycerol enrichment in plasma with a new derivative and positive-ion chemical ionization (PCI) mass spectrometry. It could be demonstrated that the trisacetyl[1,1,2,3,3-H-2(5)]glycerol derivative was able to produce a fragment at mit = 164 with sufficient intensity. Application of [1,1,2,3,3-H-2(5)]glycerol in seven healthy volunteers resulted in reproducible measurements of basal glycerol turnover rates. The mean glycerol flux rate of 3.02 +/- 0.37 mu mol.kg(-1) body wt.min(-1) after an overnight fast was similar to values reported from studies with comparable protocols. Physiological changes of lipolysis rates after 48 h of fasting followed by infusion of 4 mg.kg(-1) body wt.min(-1) glucose could also be adequately studied in one subject. Fast-induced elevated glycerol turnover at 7.56 mu mol.kg(-1) body wt.min(-1), was substantially suppressed to 1.13 mu mol.kg(-1) body wt.min(-1), when glucose was administered. The easily performed trisacetyl[1,l,2,3,3-H-2(5)]glycerol derivative analyzed by PCI mass spectrometry is suitable for studying glycerol kinetics in humans.