20(S)-protopanaxadiol (aPPD), a gut microflora metabolite of ginseng saponin, is currently undergoing early clinical trials as an antidepressant agent. As a gastrointestinal metabolite, aPPD must first undergo absorption and metabolism in the intestinal epithelium before further metabolism in the liver. The present study identifies and compares the metabolism profile and enzyme kinetics of aPPD in human intestinal microsomes (HIM) and human liver microsomes (HLM). Pooled HIM and HLM of mixed gender were obtained from XenoTech, LLC (Lenexa, KS). aPPD (22 µM) was incubated with HIM (1 mg/ml) and HLM (0.25 mg/ml) for 60 min and 10 min, respectively, to profile the metabolite formation. In vitro reaction mixtures for enzyme kinetic studies contained 67 mM potassium phosphate buffer (pH 7.4), HIM (0.25 mg/ml) or HLM (0.15 mg/ml), NADPH-regenerating system and varying concentrations of aPPD (0-33 µM) in a final volume of 0.1 ml incubated for 10 min. A liquid chromatography-matrix-assisted laser desorption/ionization time-of-flight (LC-MALDI-TOF) mass spectrometry in positive ionization mode was used for metabolite identification study, whereas for enzyme kinetic studies of metabolite formation and substrate depletion a Waters Acquity Ultraperformance liquid chromatography (UPLC) system coupled to a Quattro Premier XE triple quadrupole mass spectrometer (MS) was used to resolve the hydroxy metabolites of aPPD and the parent molecule on a BEH MS C18 column (1.7 µm, 2.1x100mm). Three major monohydroxylated metabolites (M1, M2 and M3) and five minor dihydroxylated metabolites were identified in both HIM and HLM incubation mixtures. Monohydroxylated metabolites of aPPD were identified by multiple reaction monitoring of the m/z 477>459 and 459>441 transitions, whereas m/z 443>425 and 425>123 transitions were used to detect aPPD in the LC/MS assay. MASSLYNX version 4.1 software was used for data acquisition and Quantlynx analyses. The incubation conditions, e.g. amount of protein and incubation time, were optimized for linearity of aPPD substrate depletion and monohydroxy metabolite formation. Enzyme kinetic parameters (Km and Vmax) were determined using nonlinear regression analysis in SigmaPlot Enzyme Kinetics Module (version 1.3) and were evaluated by substrate inhibition or Hill equations. The Km values for the monohydroxy metabolite formation were in the range of 15 µM to 39 µM. M1 and M2 have higher Km values in HLM than in HIM while the opposite holds for M3. Results from the present study allow us to compare the metabolite formation profile and enzyme kinetics of aPPD in two major xenobiotic metabolizing sites and provides an insight into the metabolic fate of aPPD in humans.