The chiral metabolites in human urine were investigated after ingestion of a 1,8-cineole (eucalyptol)-containing entero-coated capsule (Soledum). For identification of the various enantiomers the enantiomerically pure (−/+)-α2-hydroxy-1,8-cineole, (−/+)-β2-hydroxy-1,8-cineole, (−/+)-9-hydroxy-1,8-cineole, and (−/+)-2-oxo-1,8-cineole were prepared. To achieve this aim, after acetylation of the synthesized racemic 2- and 9-hydroxy-1,8-cineoles, pig liver esterase- or yeast-mediated hydrolysis provided the (−)-alcohols with their antipodal (+)-acetates with enantiomeric excess of 33–100 %. Dess–Martin periodinane oxidation of the alcohol (+)-α2-hydroxy-1,8-cineole, obtained by hydrolysis of the resolved acetate, provided the corresponding (+)-2-oxo-1,8-cineole, meanwhile the oxidation of (−)-α2-hydroxy-1,8-cineole gave (−)-2-oxo-1,8-cineole. Using these standards seven metabolites (+/−)-α2-hydroxy-1,8-cineole, (+/−)-β2-hydroxy-1,8-cineole, (+/−)-α3-hydroxycineole, (+/−)-3-oxo-1,8-cineole, 4-hydroxy-1,8-cineole, 7-hydroxy-1,8-cineole, and (+/−)-9-hydroxy-1,8-cineole, all liberated from their glucuronides, were identified in urine by GC-MS on a chiral stationary phase after consumption of 100 mg of 1,8-cineole. Metabolite screening using 2H3-1,8-cineol as the internal standard revealed (+/−)-α2-hydroxy-1,8-cineole as the predominant metabolite followed by (+/−)-9-hydroxy-1,8-cineole. Furthermore, the results showed that one enantiomer is always formed preferentially.