Since the 1960s, glucocorticoids are used by athletes to improve their performances. Their use is restricted in sports. Hair can document chronic abuse and can be therefore a complementary matrix for doping control. We have developed a new extraction, purification, and separation technique using liquid chromatography and mass spectrometry for the identification and quantification of two endogenous glucocorticoids: cortisol and cortisone. Qualitative and quantitative investigations were achieved with 44 hair samples (17 males, 27 females; age ranging from 2 to 90 years). Hair strands were washed in methylene chloride, the first two centimeters of the strand were cut and pulverized in a ball mill. The powdered hair was incubated in 2 mL Soerensen buffer, pH 7.6, for 16 h at 40 degrees C, in the presence of cortisol-d3 as an internal standard. Purification of the incubation medium was achieved on SPE C18 Isolute extraction columns followed by an alkaline liquid-liquid extraction with diethylether. The eluate was evaporated to dryness and resuspended in 25 microL of acetonitrile/ammonium formiate (1:1,v/v). The chromatography was operated on a LC Packings Superba Nucleosil C18 column using a linear gradient of acetonitrile from 30% to 70% in 10 min. The detector was a Perkin Elmer Sciex API 100 mass spectrometer. The detector's response was linear for cortisol and cortisone concentrations ranging from 1 to 500 pg/mg. Extraction recovery at 50 pg/mg was 74% for cortisol and 32% for cortisone. Repeatability (CV values n = 8) at 7 pg/mg cortisol and at 50 pg/mg cortisone were 11% in both cases. Limit of detection and limit of quantification were 1 and 5 pg/mg, for both compounds, respectively. Cortisol concentrations in hair ranged from 5 to 91 pg/mg (mean 18 pg/mg). Cortisone concentrations in hair ranged from 12 to 163 pg/mg (mean 70 pg/mg). No influence of hair colour could be found. Influence of sex on cortisone concentrations seems possible but could not be statistically demonstrated. Finally, cortisone concentrations in hair are significantly higher before the age of 20. Incorporation of cortisol and cortisone in hair could follow a passive diffusion through sweat after conversion of part of cortisol to cortisone by Type 2 11-beta-Hydroxysteroid-dehydrogenase in sweat glands. This issue was documented by these analyses.