The simultaneous determination of the enantiomers of the beta1-selective adrenergic antagonist atenolol in human plasma and urine is described. After an alkaline pre-extraction atenolol is extracted from biological material at pH 12.3 using dichloromethane/propan-2-ol. The separation of the underivatized enantiomers is achieved by high-performance liquid chromatography on a chiral stationary phase (Chiralcel OD, cellulose tris-3,5-dimethylphenylcarbamate, coated on silica gel) with fluorimetric detection. (--)-)S)-Pindolol is used as an internal standard. The detection limits of 5 ng/ml enantiomer in plasma and 50 ng/ml enantiomer in urine are sufficient for pharmacokinetic studies after therapeutic doses.