A liquid chromatographic-mass spectrometric method for the determination of cortisol in serum using atmospheric pressure electrospray ionization and tandem mass spectrometry is described. During sample preparation, 150 mul of serum were deproteinized with methanol/zinc sulfate followed by on-line solid phase extraction employing column switching. Tri-deuterated cortisol was used as the internal standard. The following transitions were monitored: cortisol, 363 > 309 m/z; d3-cortisol, 366 > 312 m/z. The total run-time was 5 minutes. The method proved linear (0-500 mug/l; r=0.999), precise (total coefficient of variation between 5.0% and 3.2% at a mean cortisol concentration of 15.1 mug/l and 269 mug/l, respectively; n=16) and specific with regard to relevant endogenous and exogenous steroids.